Caspase-14, an apoptotic protease, nucleic acids encoding and methods of use

ABSTRACT

The invention relates to an isolated nucleic acid molecule encoding a caspase-14 polypeptide or functional fragment thereof, a vector that contains the nucleic acid molecule and a host cell that contains the vector. The invention also relates to an isolated gene encoding caspase-14, as well as functional fragments thereof. The gene or nucleic acid molecule can include single or double stranded nucleic acids corresponding to coding or non-coding strands of the caspase-14 nucleotide sequence. Isolated caspase-14 polypeptides or functional fragments thereof are also provided, as are antibodies that specifically bind thereto. In addition, the invention relates to methods of identifying compounds that modulate caspase-14 activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.09/187,789, filed Nov. 6, 1998, which issued as U.S. Pat. No. 6,340,740on Jan. 22, 2002; which application is a continuation-in-part of U.S.application Ser. No. 09/139,600, filed Aug. 25, 1998, which issued asU.S. Pat. No. 6,432,628; which application claims the benefit ofpriority from U.S. Provisional Application No. 60/056,986, filed on Aug.26, 1997, now abandoned.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant AI 35035-1from the National Institutes of Health. Accordingly, the government hascertain rights to this invention.

TECHNICAL FIELD

This invention relates generally to caspase-14, and in particular, tonucleic acids encoding caspase-14, the encoded polypeptides, antibodiesthereto, and methods of identifying modulators of caspase-14 activity.

BACKGROUND OF THE INVENTION

Apoptosis, also referred to as physiological cell death or programmedcell death, is a normal physiological process of cell death that plays acritical role in the regulation of tissue homeostasis by ensuring thatthe rate of new cell accumulation produced by cell division is offset bya commensurate rate of cell loss due to death. Apoptosis can becharacterized by morphological changes in the cell, includingfragmentation of nuclear chromatin, compaction of cytoplasmicorganelles, dilatation of the endoplasmic reticulum, a decrease in cellvolume and alterations to the plasma membrane, resulting in therecognition and phagocytosis of apoptotic cells and prevention of aninflammatory response. Disturbances in apoptosis that prevent or delaynormal cell turnover can be just as important to the pathogenesis ofdiseases as are known abnormalities in the regulation of proliferationand the cell cycle. Like cell division, which is controlled throughcomplex interactions between cell cycle regulatory proteins, apoptosisis similarly regulated under normal circumstances by the interaction ofgene products that either induce or inhibit cell death.

The stimuli that regulate the function of these apoptotic gene productsinclude both extracellular and intracellular signals. Either thepresence or the removal of a particular stimulus can be sufficient toevoke a positive or negative apoptotic signal. Physiological stimulithat inhibit or reduce the likelihood of apoptosis include, for example,growth factors, extracellular matrix, CD40 ligand, viral gene products,neutral amino acids, zinc, estrogen and androgens. In contrast, stimulithat promote apoptosis include, for example, tumor necrosis factor(TNF), Fas, transforming growth factor β (TGFβ), neurotransmitters,growth factor withdrawal, loss of extracellular matrix attachment,intracellular calcium and glucocorticoids. Other stimuli, includingthose of environmental and pathogenic origin, also exist and can eitherinduce or inhibit apoptosis. Although apoptosis is mediated by diversesignals and complex interactions of cellular gene products, the resultsof these interactions ultimately lead into a cell death pathway that isevolutionarily conserved between humans and invertebrates.

Several gene products that modulate the apoptotic process have beenidentified. Although these products can, in general, be separated intotwo basic categories, gene products from each category can function toeither inhibit or induce apoptosis. One family of gene products is theBcl-2 family of proteins. Bcl-2 is the best characterized member of thisfamily and inhibits apoptosis when overexpressed in cells. Other membersof the Bcl-2 family of proteins include, for example, Bax, Bak,Bcl-x_(L), Bcl-x_(S) and Bad. While some of these proteins can inhibitapoptosis, others can induce apoptosis (for example, Bcl-x_(S) and Bak,respectively).

A second family of gene products, the caspase family, is relatedgenetically to the C. elegans ced-3 gene product, which is required forapoptosis in the roundworm, C. elegans. The caspase family includes, forexample, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5,caspase-6, caspase-7, caspase-8, caspase-9 and caspase-10. Among thecommon features of the caspase gene products is that 1) they arecysteine proteases with specificity for substrate cleavage at Asp-Xbonds, where “X” is an amino acid; 2) they share a conservedpentapeptide sequence within the active site; and 3) they aresynthesized as proenzymes that require proteolytic cleavage at specificaspartate residues for activation of protease activity. Cleavage of theproenzyme produces two polypeptide protease subunits, which combinenon-covalently to form a tetramer comprised of two heterodimers.Although these proteases, when expressed in cells, induce apoptosis,several alternative structural forms of these proteases, such ascaspase-1δ (ICEδ), caspase-1ε (ICEε), caspase-2_(S) (ICH-1_(S)),caspase-6β (Mch2β) and caspase-7β (Mch3β), inhibit apoptosis.

In addition to the Bcl-2 and caspase families, which play a role inapoptosis in mammalian cells, other gene products are important inmammalian apoptosis. For example, in addition to ced-3, another C.elegans gene product, ced-4, is required for apoptosis in C. elegans.Apaf-1, a human protein homologous to ced-4, binds cytochrome c and mayactivate caspase-3, leading to apoptosis. In addition, another protein,casper, while not a caspase, has sequence similarity to caspase-8throughout its length and interacts with caspase-8 and caspase-3 throughdistinct domains. Overexpression of casper in mammalian cells inducesapoptosis.

It is uncertain whether other genes encode members of either of theBcl-2 or caspase gene families and, if so, what role they play in theapoptotic pathway. It also is unclear what physiological controlmechanisms regulate apoptosis and how the apoptotic pathways interactwith other physiological processes. For example, it has been suggestedthat cytotoxic T lymphocytes mediate their destructive function byinducing apoptosis in their target cells.

The process of apoptosis maintains tissue homeostasis in variousphysiological processes, including embryonic development, immune cellregulation and normal cell turnover. It follows that the loss ofapoptosis can lead to a variety of pathological disease states. Forexample, the inappropriate loss of apoptosis can lead to thepathological accumulation of self-reactive lymphocytes such as thoseoccurring in association with many autoimmune diseases. Inappropriateloss of apoptosis also can lead to the accumulation of virally infectedcells and of hyperproliferative cells such as tumor cells. Similarly,the inappropriate activation of apoptosis can contribute to a variety ofpathological disease states including, for example, acquiredimmunodeficiency syndrome (AIDS), neurodegenerative diseases andischemic injury. Treatments that are specifically designed to modulatethe apoptotic pathways in these and other pathological conditions canchange the natural progression of many of these diseases.

Thus, there exists a need to identify apoptotic genes and their geneproducts and for methods of modulating apoptosis for the therapeutictreatment of human diseases. The present invention satisfies this needand provides related advantages as well.

SUMMARY OF THE INVENTION

The invention generally provides caspase-14. In one aspect, theinvention provides an isolated nucleic acid molecule encoding acaspase-14 polypeptide or a functional fragment thereof. Nucleic acidand amino acid sequences of caspase-14 are provided. The invention alsoprovides caspase-14 polypeptides or a functional fragment thereof.

In another aspect, a vector that contains the nucleic acid molecule anda host cell that contains the vector is also provided. Also provided isan expression vector comprising the nucleic acid molecule encodingcaspase-14 that is operatively linked to a promoter.

In other aspects, an isolated caspase-14 polypeptide and functionalfragment thereof are also provided, as are antibodies that specificallybind thereto. In addition, the invention provides methods of identifyingcompounds that modulate caspase-14 activity comprising: (a) contacting asample containing a caspase-14 polypeptide or functional fragmentthereof with a test compound, and thereafter (b) determining theactivity of caspase-14 polypeptide or functional fragment thereof.

Methods are also provided for identifying inhibitors and enhancers ofcaspase-14 activity, comprising: (a) contacting an activated caspase-14polypeptide with a substrate in the presence of a test compound underconditions in which the caspase-14 processes the substrate in theabsence of the test compound; and thereafter (b) detecting increased ordecreased substrate turnover, wherein increased substrate turnoverindicates the presence of an enhancer and wherein decreased substrateturnover indicates the presence of an inhibitor.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, the various references set forth below thatdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated herein, by reference, intheir entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the nucleotide sequence (SEQ ID NO:1 and SEQ ID NO:76for complement strand) and deduced amino acid sequence (SEQ ID NO:2) ofcaspase-14. The double-stranded nucleotide sequence is shown, withnucleotide position numbers shown on the left and right sides. Theencoded amino acids are shown below each row of nucleotides, with stopcodons designated by an asterisk. The start codon, which is shown atnucleotide positions 61 to 63, was identified based on the presence of astop codon upstream of this position (nucleotide positions 49 to 51).Amino acid position numbers are shown on the right side of the sequence,with the letter “a” shown on the left side of the amino acid sequenceand the initial methionine designated position 1. The open reading frameencodes caspase-14, which is 257 amino acids in length, and ends with astop codon at nucleotide positions 832 to 834.

FIG. 2 illustrates the amino acid-sequence analysis and primarystructure of caspase-14. FIG. 2A shows a colinear alignment of thepredicted amino acid sequence of procaspase-14 with the amino acidsequence of 8 other known caspases. Noncontiguous sequences ofcaspase-14 (SEQ ID NOS:62-68), Mch5 (caspase-8; SEQ ID NOS:48-54), Mch3(caspase-7; SEQ ID NOS:35-41), Mch2 (caspase-6; SEQ ID NOS:42-47), CPP32(caspase-3; SEQ ID NOS: 27-34), ICE (caspase-1; SEQ ID NOS:21-26), andICH-1 (caspase-2; SEQ ID NOS:55-61), caspase-11 (SEQ ID NOS:10-15),caspase-12 (SEQ ID NOS:16-20) are shown. The amino acid position of thefirst amino acid shown in the respective proteins is indicated on theleft. FIG. 2B depicts the primary structure of procaspase-14 representedby a bar diagram. The active site QACRG (SEQ ID NO:3) pentapeptide andpotential aspartate processing sites are indicated.

FIG. 3 is a scanned image of an autoradiogram representing a Northernblot of the tissue distribution in mouse of caspase-14 mRNA.

FIG. 4 is a bar diagram representing the ability of procaspase-14overexpression in MCF-7 cells to initiate apoptosis.

FIGS. 5A-C are scanned images of autoradiograms representing SDS-PAGEanalysis of the expression and processing of procaspase-14.

FIG. 6 is a scanned image of an autoradiogram representing SDS-PAGEanalysis of the processing of procaspase-14 in S-100 extracts.

FIG. 7 illustrates the nucleotide sequence (SEQ ID NO:4) and the deducedamino acid sequence (SEQ ID NO:5) of caspase-14. The nucleotide sequenceis shown, with nucleotide position numbers shown on the right side. Theencoded amino acids are shown below each row of nucleotides, with stopcodons designated by an asterisk. The start codon, which is shown atnucleotide positions 49 to 51. Amino acid position numbers are shownbelow the sequence and the initial methionine designated position 1. Theopen reading frame encodes a caspase-14, which is 242 amino acids inlength, and ends with a stop codon at nucleotide positions 775-777.

FIG. 8 illustrates the nucleotide sequence (SEQ ID NO:6) and the deducedamino acid sequence (SEQ ID NO:7) of a splice variant of humancaspase-14. The nucleotide sequence is shown, with nucleotide positionnumbers shown to the right side. The encoded amino acids are shown beloweach row of nucleotides, with stop codon designated by an asterisk. Thestart codon, which is shown at nucleotide positions 49 to 51. Amino acidposition numbers are shown below the sequence and the initial methioninedesignated position 1. The open reading frame encodes a caspase-14,which is 230 amino acids in length. The active site is at amino acidpositions 130-134 and the cleavage between the large and small subunitis at positions 146 and 147. This sequence differs from that in FIG. 7in that there is an intronic insertion at position 568 which results ina shift in the reading frame and a shorter protein.

FIG. 9 illustrates the nucleotide sequence (SEQ ID NO:8) and the deducedamino acid sequence (SEQ ID NO:9) of a splice variant of humancaspase-14. The nucleotide sequence is shown, with nucleotide positionnumbers shown to the right side. The encoded amino acids are shown beloweach row of nucleotides, with stop codon designated by an asterisk. Thestart codon, which is shown at nucleotide positions 49 to 51. Amino acidposition numbers are shown below the sequence and the initial methioninedesignated position 1. The open reading frame encodes a caspase-14,which is 214 amino acids in length. The active site is at amino acidpositions 102-106 and the cleavage site separating the large and smallsubunit is between positions 118 and 119. This sequence differs fromthat in FIG. 7 in that it has an internal deletion at position 151,which results in a shorter protein.

FIG. 10 is an identity comparison between the mouse (SEQ ID NO:2) andhuman (SEQ ID NO:5) caspase-14 polypeptide sequences as described inExample 1. The mouse sequence is represented on the top line and thehuman sequence is represented on the bottom line.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a cell death specific protease, termedcaspase-14, which is a member of the caspase family of proteases thatincludes, for example, with alternate designations in parentheses,caspase-1 (ICE, interleukin-1-β converting enzyme), caspase-2_(L)(ICH-1_(L)), caspase-2_(S) (ICH-1_(S)), caspase-3 (CPP32), caspase-4(TX, ICH-2, ICE_(rel)-II), caspase-5 (ICE_(rel)-III, TY), caspase-6(Mch2), caspase-7 (Mch3, ICE-LAP3, CMH-1), caspase-8 (Mch5, MACH,FLICE), caspase-9 (Mch6, ICE-LAP6) and caspase-10 (Mch4). Similar toother caspases, caspase-14 is produced as a proenzyme and becomes activefollowing proteolytic cleavage into a larger and smaller subunit. Thetwo subunits form heterodimers that associate with each other into aheterotetrameric active complex, which induces apoptosis. Substratespecificity uniquely requires an aspartic acid residue in the P1position of the substrate binding site with a small, preferablyhydrophobic, residue in the P1′ position.

A nucleic acid molecule (SEQ ID NO:1), which encodes a caspase-14polypeptide (SEQ ID NO:2) was identified and isolated based onidentifying an expressed sequence tag (EST) having GenBank accessionnumber AA103647, a sequence of 483 nucleotides in length. The EST wasidentified during a homology search of the GenBank database using aquery nucleotide sequence based on caspase-3 and caspase-6 codingsequences (see Example 1). The mouse cell clone that contained thesequence from which the EST was derived was obtained from IMAGEConsortium. It was discovered that the clone, which had been onlypartially and inaccurately sequenced, contained a nucleotide sequence(SEQ ID NO:1) encoding caspase-14. This caspase had the highest homologywith procaspase-3 (32% identity) and procaspase-7 (31% identity). Thedifferences between the EST and the corresponding sequence of the codingstrand shown in FIG. 1 (SEQ ID NO:1) include, for example, that thecoding strand shown in FIG. 1 (SEQ ID NO:1) contains a cytosine atnucleotide position 13 and guanines at nucleotide positions 54 and 164,while the EST contains nothing at the corresponding positions.

The invention also provides additional caspase-14 nucleic acid moleculessuch as (SEQ ID NO:4), that encodes human caspase-14 (SEQ ID NO:5),which is preferentially expressed in keratenocytes as determined byRT-PCR in normal keratenocytes as well as transformed keratenocytes suchas the A431 cell line. As demonstrated by FIG. 7, the nucleotidesequence is 777 nucleotides, while the encoded polypeptide is 242 aminoacids in length. This human sequence was identified as demonstrated inExample 1, using nested PCR primers corresponding to the mouse sequence(SEQ ID NO:1). Further provided are splice variant isoforms of SEQ IDNO:4. In one embodiment such isoforms are provided by nucleic acidmolecules illustrated in FIG. 8 (SEQ ID NO:6) and FIG. 9 (SEQ ID NO:8)as well as their respectively encoded human caspase-14 polypeptides (SEQID NOS:7 and 9). Such splice variants are identified using highstringency probes derived from SEQ ID NOS:1 or 4.

The invention provides isolated caspase-14 polypeptides such as SEQ IDNOS:2 or 5 as well as splice variants thereof (e.g., SEQ ID NOS:7 and9). The term “isolated” means in a form that is relatively free fromcontaminating lipids, unrelated polypeptides, nucleic acids and othercellular material normally associated with the polypeptide in the celland at least about 30% of the total material. In another embodiment ofthe invention, the isolated caspase-14 polypeptide is about 50% of thetotal material. In another embodiment of the invention, the isolatedcaspase-14 polypeptide is about 70% of the total material. In anotherembodiment of the invention, the isolated caspase-14 polypeptide isabout 90% of the total material. In yet another embodiment of theinvention, the isolated caspase-14 polypeptide is greater than about 95%of the total material. Thus, an isolated polypeptide of the invention isone that is in a form that is different from the naturally occurringstate.

Exemplary polypeptides of the invention are the isolated mousecaspase-14 polypeptide 257 amino acids in length and shown as SEQ IDNO:2 (FIG. 1), the isolated human caspase-14 polypeptide 242 amino acidsin length and shown as SEQ ID NO:5 (FIG. 7), and the isolated humancaspase-14 isoforms shown as SEQ ID NOS:7 and 9. The invention furtherprovides an isolated caspase-14 polypeptide, which has greater thanabout 33% amino acid sequence identity with SEQ ID NOS:2, 5 or theirrespective isoforms. In other embodiments of the invention, thepolypeptide has generally greater than about 50% or 60% amino acidsequence identity with SEQ ID NOS:2, 5 or their respective splicevariant isoforms. In yet other embodiments of the invention, thepolypeptide has generally greater than about 70% or 80% amino acidsequence identity with SEQ ID NOS:2, 5, or their respective isoforms.Such amino acid sequence identity may be determined by standardmethodologies, including use of the National Center for BiotechnologyInformation BLAST search methodology available at www.ncbi.nlm.nih.gov.The identity methodologies preferred are those described in U.S. Pat.No. 5,691,179 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997all of which are incorporated herein by reference. Most preferred andthat which is used to calculate percentages herein is the pileUPalgorithm as described in Example 1.

A caspase-14 polypeptide includes polypeptides having substitutions ofconserved and non-essential amino acids of SEQ ID NOS:2 or 5 and,generally includes, for example, mammalian homologues of SEQ ID NOS:2 or5 such as rat or other mammalian caspase-14. A caspase-14 polypeptidealso can include polypeptides having related but different sequences,provided the polypeptide has at least one functional activity of SEQ IDNOS:2 or 5, such as protease activity. For example, splice variantisoforms of SEQ ID NOS:1 or 4, such as those provided by SEQ ID NOS:6and 8 are included in such a definition. Therefore, it should beunderstood that when referencing the various polypeptides and nucleicacid molecules, the splice variant isoforms and homologous sequencesthereof are implicated as well. Accordingly, with regard to fragments,the specific disclaimers to contiguous sequences found in the prior artas to SEQ ID NOS:1, 2, 4, and 5, also includes the identical sequencesfound in SEQ ID NOS:6-9.

It is understood that limited modifications may be made to a caspase-14polypeptide without destroying its biological function and that only aportion of the entire primary structure may be required in order toeffect activity. Thus, for example, minor modifications of SEQ ID NOS:2or 5 provide examples of caspase-14 polypeptides. Such minormodifications may result in polypeptides that have substantiallyequivalent or enhanced function as compared to SEQ ID NOS:2 or 5. Thesemodifications may be deliberate, such as through site-directedmutagenesis, or may be accidental, such as through mutation in hoststhat are caspase-14 producers. It also is understood that allelicvariants and splice variants of caspase-14 are caspase-14 polypeptidesencompassed within the invention.

In addition, the invention provides a functional fragment of SEQ IDNOS:2 or 5 or splice variants thereof. A functional fragment of SEQ IDNOS:2 or 5 is defined structurally and functionally in that it has thesame contiguous sequence as a portion of SEQ ID NOS:2 or 5 and at leastone biological activity characteristic of caspase-14. A functionalfragment of SEQ ID NOS:2, 5, or a splice variant thereof comprises atleast 8 contiguous residues of SEQ ID NOS:2, 5, or a splice variantthereof. In other embodiments of the invention, a functional fragment ofSEQ ID NOS:2, 5, or a splice variant thereof, comprises an amino acidsequence of at least 10 or 12 contiguous residues. In other embodimentsof the invention, a functional fragment of SEQ ID NOS:2 or 5 comprisesan amino acid sequence of at least 15 or 20 contiguous residues. Inother embodiments of the invention, a functional fragment of SEQ IDNOS:2, 5, or splice variants thereof, comprises an amino acid sequenceof at least 25 or 30 contiguous residues. In another embodiment of theinvention, a functional fragment of SEQ ID NOS:2, 5, or splice variantsthereof comprises an amino acid sequence of at least 50 contiguousresidues. In yet other embodiments of the invention, a functionalfragment of SEQ ID NO:2 comprises an amino acid sequence of at least 6or 7 contiguous residues of SEQ ID NO:2, provided that such sequencedoes not include amino acid positions 132 to 138 or 134 to 139 of SEQ IDNO:2 or the homologous sequences from splice variant forms. In yet otherembodiments of the invention, a functional fragment of SEQ ID NO:5comprises an amino acid sequence of at least 6 or 7 contiguous residuesof SEQ ID NO:2, provided that such sequence does not include amino acidpositions 128 to 133 or 130 to 135 of SEQ ID NO:5 or the homologoussequences from splice variant forms. However, an amino acid sequencethat consists of the identical amino acid sequence encoded by the ESThaving GenBank accession number AA103647, or any contiguous portionthereof, is not a functional fragment of SEQ ID NOS:2 or 5 encompassedwithin the invention. Similarly, a contiguous portion of caspase-1,caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7,caspase-8, caspase-9, caspase-10 or ced-3 is not a functional fragmentof SEQ ID NOS:2, 5, 7, or 9, encompassed within the invention. Abiological activity of a functional fragment of SEQ ID NOS:2 or 5 is anactivity of caspase-14 and can be, for example, the ability to bind aligand, have protease or other enzymatic activity, enhance or inhibitapoptosis or bind or induce the production of an anti-caspase-14antibody.

The invention also provides a functional fragment of a caspase-14polypeptide. Such a functional fragment is defined structurally andfunctionally in that it has amino acid sequence identity to a portion ofSEQ ID NOS:2 or 5, as described below, and has at least one biologicalactivity of caspase-14, as described above. A functional fragment of acaspase-14 polypeptide that does not include QACRG (SEQ ID NO:3; aminoacid positions 134 to 138 of SEQ ID NO:2; amino acid positions 130-134of SEQ ID NO:5 or the homologous portions of a splice variant) hasseveral embodiments. In one embodiment such a functional fragmentcomprises at least 10 amino acids and has at least about 70% amino acidsequence identity with a portion of SEQ ID NOS:2, 5, 7, or 9. In otherembodiments, such a functional fragment comprises at least 10 aminoacids and has at least about 75% or 80% amino acid sequence identitywith a portion of SEQ ID NOS:2, 5, 7, or 9. In other embodiments, such afunctional fragment comprises at least 10 amino acids and has at leastabout 85% or 90% amino acid sequence identity with a portion of SEQ IDNOS:2, 5, 7, or 9. In another embodiment, such a functional fragmentcomprises at least 25 amino acids and has at least about 65% amino acidsequence identity with a portion of SEQ ID NOS:2, 5, 7, or 9. In otherembodiments, such a functional fragment comprises at least 25 aminoacids and has at least about 70% or 75% amino acid sequence identitywith a portion of SEQ ID NOS:2, 5, 7 or 9. In other embodiments, such afunctional fragment comprises at least 25 amino acids and has at leastabout 80% or 85% amino acid sequence identity with a portion of SEQ IDNOS:2, 5, 7, or 9. In other embodiments, such a functional fragmentcomprises at least 40 amino acids and has at least about 50% or 60%amino acid sequence identity with a portion of SEQ ID NOS:2, 5, 7, or 9.In yet other embodiments, such a functional fragment comprises at least40 amino acids and has at least about 70% or 80% amino acid sequenceidentity with a portion of SEQ ID NOS:2, 5, 7, or 9. The aforementionedidentities being calculated with the pileUP algorithm as defined inExample 1.

In comparison, a functional fragment of a caspase-14 polypeptide thatincludes QACRG (SEQ ID NO:3; amino acid positions 134 to 138 of SEQ IDNO:2; amino acid positions 130 to 134 of SEQ ID NO:5 or homologousportions of splice variants) comprises at least about 13 amino acids andhas greater than about 92% amino acid sequence identity with a portionof SEQ ID NOS:2, 5, 7, or 9. In other embodiments of the invention, sucha functional fragment comprises at least about 13 amino acids and hasgreater than about 93% or 95% amino acid sequence identity with aportion of SEQ ID NOS:2, 5, 7, or 9. In another embodiment of theinvention, such a functional fragment comprises at least about 13 aminoacids and has greater than about 98% amino acid sequence identity with aportion of SEQ ID NOS:2, 5, 7, or 9. In another embodiment of theinvention, such a functional fragment comprises at least about 25 aminoacids and has greater than about 72% amino acid sequence identity with aportion of SEQ ID NOS:2, 5, 7 or 9. In other embodiments of theinvention, such a functional fragment or comprises at least about 25amino acids and has greater than about 75% or 80% amino acid sequenceidentity with a portion of SEQ ID NOS:2, 5, 7, or 9. In yet otherembodiments of the invention, such a functional fragment or comprises atleast about 25 amino acids and has greater than about 85% or 90% or 95%amino acid sequence identity with a portion of SEQ ID NOS:2, 5, 7, or 9.A fragment that consists of the identical amino acid sequence encoded bythe EST having GenBank accession number AA103647, or any contiguousportion thereof, or of any contiguous portion of caspase-1, caspase-2,caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8,caspase-9, caspase-10 or ced-3, is not considered a functional fragmentof a caspase-14 polypeptide.

It is understood that functional fragments of a caspase-14 polypeptideinclude fragments with substitutions of conserved and non-essentialamino acids of portions of SEQ ID NO:2 and, therefore, include, forexample, fragments of eukaryotic homologs of SEQ ID NO:2 such asfragments of yeast or Drosophila or C. elegans caspase-14. However, italso is understood, that contiguous fragments of caspase-1, caspase-2,caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8,caspase-9, caspase-10 or ced-3 polypeptides, for example, are notfunctional fragments of a caspase-14 polypeptide encompassed within theinvention.

SEQ ID NOS:2 or 5 are inactive proenzymes, which are proteolyticallycleaved to form a large subunit and a small subunit, which provideexamples of functional fragments of SEQ ID NOS:2 or 5. Proteolyticcleavage of SEQ ID NO:2 occurs between the aspartic acid and glutamicacid residues of the shown in FIG. 2. Thus, cleavage occurs betweenamino acid positions 156 and 157 of SEQ ID NO:2 and between amino acidpositions 162 and 163 of SEQ ID NO:2, resulting in a large subunitcomprising amino acid positions 1 to 156 of SEQ ID NO:2 and a smallsubunit comprising amino acid positions 163 to 257 of SEQ ID NO:2.Proteolytic cleavage of SEQ ID NO:5 occurs between the aspartic acid andglutamic acid residues at positions 146 and 147 of SEQ ID NO:5,resulting in a large subunit comprising amino acids 1-146 of SEQ ID NO:5and a small subunit comprising amino acid positions 147-242 of SEQ IDNO:5. Other caspase-14 polypeptides, which are inactive proenzymes, alsoare proteolytically cleaved to form large subunits and small subunits,which provide examples of functional fragments of caspase-14. The largeand small subunits of a caspase-14 polypeptide can combinenon-covalently to produce a heterotetramer having apoptotic activity.

If desired, the large subunit of a caspase-14 polypeptide can becombined with a small subunit of another caspase polypeptide such ascaspase-3 (CPP32) to form an apoptotic complex, or the small subunit ofa caspase-14 polypeptide can be combined with a large subunit of anothercaspase protein such as caspase-3 (CPP32) to form an apoptotic complex.Such complexes can be formed in vitro, in cells in culture, or in vivoby heterodimerization of the large and small subunits.

The activity of a caspase-14 polypeptide or functional fragment thereofcan be measured enzymatically (see Example 2). If desired, a caspase-14polypeptide or functional fragment thereof can be attached to a secondmolecule such as, for example, a protein, carbohydrate, lipid orchemical moiety. For example, a caspase-14 polypeptide or functionalfragment thereof can be fused to a heterologous protein such as a fusionprotein that retains caspase-14 enzymatic or other biological activityand has a characteristic of the heterologous protein.

An isolated caspase-14 polypeptide or functional fragment thereof can beobtained by a variety of methods known in the art. For example, acaspase-14 polypeptide can be isolated by biochemical methods such asaffinity chromatography. Affinity matrices that can be used forcaspase-14 isolation can be a solid phase having attached theretoanti-caspase-14 monoclonal or polyclonal antibodies prepared against acaspase-14 polypeptide or a functional fragment thereof comprising acaspase-14 epitope. Alternatively, ligands such as substrate analoguesor enzymatic inhibitors of caspase-14 can be used as affinity matricesto isolate a caspase-14 polypeptide or functional fragment thereof thatbinds the ligand.

Other biochemical methods for isolating a caspase-14 polypeptide orfunctional fragment thereof include preparative gel electrophoresis, gelfiltration, affinity chromatography, ion exchange and reversed phasechromatography, chromatofocusing, isoelectric focusing and sucrose orglycerol density gradients (Deutscher, Methods in Enzymology: Guide toProtein Purification, Vol. 182, Academic Press, Inc., San Diego (1990),Chapter 38; Balch et al., Methods in Enzymology, Vol. 257, AcademicPress, Inc., San Diego (1995), Chapter 8). For example, a caspase-14polypeptide or functional fragment thereof can be isolated bypreparative polyacrylamide gel electrophoresis and elution by diffusionor electroelution (Deutscher, supra, 1990, Chapter 33). Continuouselution gel electrophoresis using a system such as the Model 491 PrepCell (BioRad, Hercules, Calif.) can be used to isolate a caspase-14polypeptide or functional fragment thereof. If desired, continuouselution gel electrophoresis can be combined with further purificationsteps such as liquid phase preparative isoelectric focusing using, forexample, the Rotofor system (BioRad).

A caspase-14 polypeptide or functional fragment thereof also can beproduced by chemical synthesis, for example, by the solid phase peptidesynthesis method (Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964)).Standard solution methods well known in the art also can be used tosynthesize a caspase-14 polypeptide or functional fragment thereof(Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, Berlin(1984); Bodanszky, Peptide Chemistry, Springer-Verlag, Berlin (1993)). Anewly synthesized caspase-14 polypeptide or functional fragment thereofcan be isolated, for example, by high performance liquid chromatographyand can be characterized using mass spectrometry or amino acid sequenceanalysis.

A caspase-14 polypeptide or functional fragment thereof also can beproduced by recombinant DNA methods. Accordingly, the invention providesa nucleic acid molecule encoding a caspase-14 polypeptide or functionalfragment thereof. Such a nucleic acid molecule can be cloned into anappropriate vector for propagation, manipulation or expression asdesired. Such a vector is commercially available or can be constructedby those skilled in the art and contains expression elements necessaryfor the transcription, translation, regulation, and, if desired, sortingof the caspase-14 polypeptide or functional fragment thereof. Theselected vector also can be used in a procaryotic or eukaryotic hostsystem, as appropriate, provided the expression and regulatory elementsare of compatible origin. A recombinant caspase-14 polypeptide orfunctional fragment thereof produced in a host cell or secreted from thecell can be isolated using, for example, an anti-caspase-14 antibody, asdescribed herein.

Caspase-14 may be expressed in a variety of host organisms. In certainembodiments, caspase-14 is produced in bacteria, such as E. coli, ormammalian cells (e.g., CHO and COS-7), for which many expression vectorshave been developed and are available. Other suitable host organismsinclude other bacterial species, and eukaryotes, such as yeast (e.g.Saccharomyces cerevisiae), and insect cells (e.g., Sf9).

In one embodiment, a DNA sequence encoding caspase-14 is introduced intoan expression vector appropriate for the host cell. In certainembodiments, caspase-14 is inserted into a vector such that a fusionprotein is produced. The caspase-14 sequence is derived as describedherein. As discussed above, the sequence may contain alternative codonsfor each amino acid with multiple codons. The alternative codons can bechosen as “optimal” for the host species. Restriction sites aretypically incorporated into the primer sequences and are chosen withregard to the cloning site of the vector. If necessary, translationalinitiation and termination codons can be engineered into the primersequences.

At a minimum, the vector will contain a promoter sequence. As usedherein, a “promoter” refers to a nucleotide sequence that containselements that direct the transcription of a linked gene. At a minimum, apromoter contains an RNA polymerase binding site. More typically, ineukaryotes, promoter sequences contain binding sites for othertranscriptional factors that control the rate and timing of geneexpression. Such sites include TATA box, CAAT box, POU box, AP1 bindingsite, and the like. Promoter regions may also contain enhancer elements.When a promoter is linked to a gene so as to enable transcription of thegene, it is “operatively linked”.

Other regulatory sequences may be included. Such sequences include atranscription termination sequence, secretion signal sequence, origin ofreplication, selectable marker, and the like. The regulatory sequencesare operationally associated with one another to allow transcription ortranslation.

The expression vectors used herein include a promoter designed forexpression of the proteins in a host cell (e.g., bacterial). Suitablepromoters are widely available and are well known in the art. Inducibleor constitutive promoters are preferred. Such promoters for expressionin bacteria include promoters from the T7 phage and other phages, suchas T3, T5, and SP6, and the trp, lpp, and lac operons. Hybrid promoters(see, U.S. Pat. No. 4,551,433), such as tac and trc, may also be used.Promoters for expression in eukaryotic cells include the P10 orpolyhedron gene promoter of baculovirus/insect cell expression systems(see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051,and 5,169,784), MMTV LTR, CMV IE promoter, RSV LTR, SV40,metallothionein promoter (see, e.g., U.S. Pat. No. 4,870,009), ecdysoneresponse element system, tetracycline-reversible silencing system(tet-on, tet-off), and the like.

The promoter controlling transcription of caspase-14 may itself becontrolled by a repressor. In some systems, the promoter can bederepressed by altering the physiological conditions of the cell, forexample, by the addition of a molecule that competitively binds therepressor, or by altering the temperature of the growth media. Preferredrepressor proteins include, but are not limited to the E. coli lacIrepressor responsive to IPTG induction, the temperature sensitive λcI857repressor, and the like.

In other optional embodiments, the vector also includes a transcriptiontermination sequence. A “transcription terminator region” has either asequence that provides a signal that terminates transcription by thepolymerase that recognizes the selected promoter and/or a signalsequence for polyadenylation.

In one aspect, the vector is capable of replication in the host cells.Thus, when the host cell is a bacterium, the vector preferably containsa bacterial origin of replication. Bacterial origins of replicationinclude the f1-ori and col E1 origins of replication, especially the oriderived from pUC plasmids. In yeast, ARS or CEN sequences can be used toassure replication. A well-used system in mammalian cells is SV40 ori.

The plasmids also preferably include at least one selectable marker thatis functional in the host. A selectable marker gene includes any genethat confers a phenotype on the host that allows transformed cells to beidentified and selectively grown. Suitable selectable marker genes forbacterial hosts include the ampicillin resistance gene (Amp^(r)),tetracycline resistance gene (Tc^(r)) and the kanamycin resistance gene(Kan^(r)). The kanamycin resistance gene is presently preferred.Suitable markers for eukaryotes usually require a complementarydeficiency in the host (e.g., thymidine kinase (tk) in tk-hosts).However, drug markers are also available (e.g., G418 resistance andhygromycin resistance).

The sequence of nucleotides encoding caspase-14 may also include asecretion signal, whereby the resulting peptide is a precursor proteinprocessed and secreted. The resulting processed protein may be recoveredfrom the periplasmic space or the fermentation medium. Secretion signalssuitable for use are widely available and are well known in the art (vonHeijne, J. Mol. Biol. 184:99-105, 1985). Prokaryotic and eukaryoticsecretion signals that are functional in E. coli (or other host) may beemployed. The presently preferred secretion signals include, but are notlimited to, those encoded by the following E. coli genes: pelB (Lei etal., J. Bacteriol. 169:4379, 1987), phoA, ompA, ompT, ompF, ompC,beta-lactamase, and alkaline phosphatase.

One skilled in the art will appreciate that there are a wide variety ofsuitable vectors for expression in bacterial cells and which are readilyobtainable. Vectors such as the pET series (Novagen, Madison, Wis.), thetac and trc series (Pharmacia, Uppsala Sweden), pTTQ18 (AmershamInternational plc, England), pACYC 177, pGEX series, and the like aresuitable for expression of caspase-14. Baculovirus vectors, such aspBlueBac (see, e.g., U.S. Pat. Nos. 5,278,050, 5,244,805, 5,243,041,5,242.687, 5,266,317, 4,745,051, and 5,169,784; available fromInvitrogen, San Diego) may be used for expression in insect cells, suchas Spodoptera frugiperda sf9 cells (see, U.S. Pat. No. 4,745,051). Thechoice of a bacterial host for the expression of caspase-14 is dictatedin part by the vector. Accordingly, commercially available vectors arepaired with suitable hosts.

A wide variety of suitable vectors for expression in eukaryotic cellsare also available. Such vectors include pCMVLacI, pXT1 (StratageneCloning Systems, La Jolla, Calif.); pCDNA series, pREP series, pEBVHis(Invitrogen, Carlsbad, Calif.). In certain embodiments, the caspase-14nucleic acid molecule is cloned into a gene targeting vector, such aspMClneo, a pOG series vector (Stratagene Cloning Systems).

Caspase-14 polypeptides may be isolated by standard methods, such asaffinity chromatography, size exclusion chromatography, metal ionchromatography, ionic exchange chromatography, HPLC, and other knownprotein isolation methods, (see generally Ausubel et al. supra; Sambrooket al. supra). An isolated purified protein gives a single band onSDS-PAGE when stained with Coomassie blue.

In another embodiment, chimeric caspases or protein fusion-caspases canbe constructed by standard molecular biological techniques as describedby Sambrook et al., supra; Ausubel et al., supra. Briefly, the region ofinterest of one caspase can be cloned into a cloning vector and with theaid of restriction enzymes digested such that the nucleic acid sequenceof another caspase may be fused thereto, thereby creating a chimericnucleic acid molecule encoding a chimeric protein. The same procedurecan be used to create a caspase fusion protein, however, in this casemany vectors are commercially available which contain fusion constructsand allow direct cloning of the insert of interest into the vector in asimple one step process.

Purified caspase-14 fusion proteins may be used in assays to screen formolecules which modulate apoptosis as described in detail infra. Infurther embodiments, these proteins may also be crystallized andsubjected to X-ray analysis to determine the 3-dimensional structure orutilized to generate antibodies.

A recombinant caspase-14 polypeptide or functional fragment thereof canbe expressed as a fusion protein with a heterologous “tag” forconvenient isolation from bacterial or mammalian host proteins. Forexample, a histidine-tagged recombinant caspase-14 polypeptide can beisolated by nickel-chelate chromatography. Similarly, aglutathione-S-transferase tag or an antigenic tag such as “FLAG,” “AU”or a myc epitope tag also can be included in a recombinant caspase-14polypeptide or functional fragment thereof (Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork (1989)). PINPOINT (Promega Corp.; Madison Wis.) is a commerciallyavailable system for expression of a caspase-14 polypeptide orfunctional fragment thereof as a fusion protein with a heterologousbiotinylated peptide.

A functional fragment of a caspase-14 polypeptide also can be produced,for example, by chemical or proteolytic cleavage of an isolatedcaspase-14 polypeptide. Methods for chemical and proteolytic cleavageand for purification of the resultant polypeptide fragments are wellknown in the art (Deutscher, supra, 1990).

A caspase-14 polypeptide or functional fragment thereof can be part of aheterodimer or a heterotetrameric apoptotic complex. Conversely, acaspase-14 inhibitor such as the large subunit of caspase-14 that lacksthe active site QACRG (SEQ ID NO:3; positions 134-138 of SEQ ID NO:2;positions 130-134 of SEQ ID NO:5), for example, can bind the smallsubunit of caspase-14 and prevent an active protease complex fromforming. Thus, a caspase-14 polypeptide or functional fragment thereofcan be screened, for example, for apoptotic activity and a caspase-14inhibitor can be screened for anti-apoptotic activity. Apoptoticactivity is the ability either alone, or in combination with anothermolecule, to produce cell death accompanied by at least one of themorphological or biochemical alterations characteristic of apoptosis.Morphological alterations characteristic of apoptosis are well known inthe art and include, for example, condensed and rounded cellularmorphology; membrane blebbing; the formation of apoptotic bodies, whichare membrane-bound bodies containing cytoplasmic and nuclear components;and condensation of the nucleus, with cytoplasmic organelles beingrelatively well maintained (Cohen, Gerald, supra, 1997; Studzinski(Ed.), Cell Growth and Apoptosis, Oxford: Oxford University Press(1995)). Biochemical alterations characteristic of apoptosis also arewell known in the art. The classical biochemical alterationcharacteristic of apoptosis is the appearance of oligonucleosome-sizedfragments of DNA, which produce a “ladder” upon agarose gelelectrophoresis. This extensive fragmentation can be preceded by anearlier endonucleolytic cleavage of chromatin, producing DNA fragmentsof about 50 kb to 300 kb in size.

A variety of assays for determining whether a caspase-14 polypeptide orfunctional fragment thereof has apoptotic activity or whether acaspase-14 inhibitor has anti-apoptotic activity are well known in theart. Such methods include light microscopy for determining the presenceof one or more morphological characteristics of apoptosis, such ascondensed or rounded morphology, shrinking and blebbing of thecytoplasm, preservation of structure of cellular organelles includingmitochondria, and condensation and margination of chromatin.

A caspase-14 polypeptide or functional fragment thereof or a caspase-14inhibitor also can be assayed for respective apoptotic or anti-apoptoticactivity using terminal deoxytransferase-mediated (TdT) dUTP biotin nickend-labeling (TUNEL) (Gavriel et al., J. Cell Biol. 119:493 (1992);Gorczyca et al., Int. J. Oncol. 1:639 (1992); Studzinski, supra, 1995).APOPTAG (ONCOR, Inc.; Gaithersburg Md.) is a commercially available kitfor identification of apoptotic cells using digoxygenin labeling. Inaddition, a caspase-14 polypeptide or functional fragment thereof or acaspase-14 inhibitor can be assayed for respective apoptotic oranti-apoptotic activity by detecting nucleosomal DNA fragments usingagarose gel electrophoresis (Studzinski, supra, 1995; Gong et al., Anal.Biochem. 218:314 (1994)).

DNA filter elution methodology also can be used to detectapoptosis-associated DNA fragmentation and to determine apoptotic oranti-apoptotic activity (Studzinski, supra, 1995: Bertrand et al., DrugDevel. 34:138 (1995)). Apoptotic or anti-apoptotic activity also can bedetected and quantitated by determining an altered mitochondrial tonuclear DNA ratio as described in Tepper et al., Anal. Biochem. 203:127(1992) and Tepper and Studzinski, J. Cell Biochem. 52:352 (1993). Oneskilled in the art understands that these, or other assays for apoptoticor anti-apoptotic activity, can be performed using routine methodology.

In another embodiment, the invention provides antibodies thatspecifically bind to caspase-14-specific epitopes. Suchcaspase-14-specific epitopes are present in caspase-14 polypeptides andfunctional fragments thereof but not in other caspase polypeptides.Antibodies that bind caspase-14-specific epitopes readily are identifiedby their inability to cross react with other caspases, ced-3 and thelike.

A caspase-14 polypeptide or functional fragment thereof can comprise animmunogenic amino acid sequence or, if haptenic, can be conjugated toanother molecule to become immunogenic, as described below. Thus, acaspase-14 polypeptide or functional fragment thereof can be useful foreliciting production of an anti-caspase-14 antibody. In addition, theinvention provides a cell line producing an anti-caspase-14 antibody.

As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. With regard to an anti-caspase-14 antibodyof the invention, the term “antigen” means a caspase-14 polypeptide or afunctional fragment thereof. An anti-caspase-14 antibody, or antigenbinding fragment of such an antibody, is characterized by havingspecific binding activity for a caspase-14-specific epitope of at leastabout 1×10⁵ M⁻¹, generally at least about 1×10⁶M⁻¹ and preferably atleast about 1×10⁸M⁻¹. Fab, F(ab′)₂, Fd and Fv fragments of ananti-caspase-14 antibody, which retain specific binding activity for acaspase-14-specific epitope, are encompassed within the anti-caspase-14antibody of the invention.

In addition, the term “antibody” as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains (Huse et al., Science246:1275-1281 (1989)). These and other methods of making, for example,chimeric, humanized, CDR-grafted, single chain, and bifunctionalantibodies are well known in the art (Winter and Harris, Immunol. Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York (1992); Borrabeck, Antibody Engineering, 2d ed., Oxford Univ.Press (1995); Hilyard et al., Protein Engineering: A practical approach,IRL Press (1992)).

An anti-caspase-14 antibody can be raised using as an immunogen such as,for example, an isolated caspase-14 polypeptide such as SEQ ID NOS:2 or5, which can be prepared from natural sources or produced recombinantly,as described above, or a functional fragment of a caspase-14polypeptide, including synthetic peptides, as described above. Anon-immunogenic peptide portion of a functional fragment of a caspase-14polypeptide can be made immunogenic by coupling the hapten to a carriermolecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH), or by expressing the peptide portion as a fusion protein. Variousother carrier molecules and methods for coupling a hapten to a carriermolecule are well known in the art (Harlow and Lane, supra, 1992).

An anti-caspase-14 antibody is useful, for example, for determining thepresence or level of caspase-14 in a tissue sample, which can be a celllysate or a histological section. The identification of the presence orlevel of caspase-14 in a sample can be made using well known immunoassayand immunohistochemical methods (Harlow and Lane, supra, 1992). Inaddition, an anti-caspase-14 antibody can be used in a screening assayto identify agents that modulate the activity of caspase-14 or thatmodulate the binding of caspase-14 to a second protein.

A particularly useful anti-caspase-14 antibody is one that binds acaspase-14 polypeptide, such as SEQ ID NOS:2 or 5, but not to either thelarge or small subunit cleavage products of the caspase-14 polypeptide,such as amino acid positions 1 to 156 and 163 to 257 of SEQ ID NO:2 or 1to 146 and 147 to 242 of SEQ ID NO:5, respectively, as well as thecorresponding large and small subunits of splice variant isoforms.Similarly, an antibody that binds to either the large subunit or thesmall subunit of a caspase-14 polypeptide, but not to the other subunitor the caspase-14 polypeptide, as well as an antibody that binds to aheterodimer comprising the large subunit and the small subunit of acaspase-14 polypeptide or a heterotetramer, but not to the caspase-14polypeptide, is useful. An antibody that binds a caspase-14 polypeptideis useful to isolate caspase-14 from a sample, whereas an antibody thatbinds the large subunit or the small subunit of a caspase-14 polypeptideis useful to identify samples with caspase-14 processing activity. Anantibody that binds a caspase-14 subunit heterodimer or heterotetrameris useful to isolate caspase-14 with apoptotic activity or in ascreening assay to identify, for example, an agent that inhibitsheterodimer or heterotetramer formation and, therefore, apoptosis. Forconvenience, reference herein to an anti-caspase-14 antibody generallyincludes all such antibodies, although the skilled artisan willrecognize that the choice of a particular antibody will depend on thepurpose for which the antibody will be used.

A kit incorporating an anti-caspase-14 antibody can be particularlyuseful. Such a kit can contain, in addition to an anti-caspase-14antibody, a reaction cocktail that provides the proper conditions forperforming the assay, control samples that contain known amounts ofcaspase-14 or other appropriate caspase-14 antigen recognized by theantibody and, if desired, a second antibody specific for theanti-caspase-14 antibody. Such an assay also can include a simple methodfor detecting the presence or amount of caspase-14 in a sample that isbound to the anti-caspase-14 antibody.

An anti-caspase-14 antibody, as well as a caspase-14 polypeptide orfunctional fragment thereof, can be labeled so as to be detectable usingmethods well known in the art (Hermanson, Bioconjugate Techniques,Academic Press (1996); Harlow and Lane, supra, 1992). For example, ananti-caspase-14 antibody can be labeled with various detectable moietiesincluding a radiolabel, an enzyme, biotin or a fluorochrome. Reagentsfor labeling an anti-caspase-14 antibody can be included in a kitcontaining the antibody or can be purchased separately from a commercialsource.

Following contact for example, of a labeled antibody with a sample suchas a tissue homogenate or a histological section of a tissue,specifically bound labeled antibody can be identified by detecting theparticular moiety. Alternatively, a labeled second antibody can be usedto identify specific binding of an unlabeled anti-caspase-14 antibody. Asecond antibody generally will be specific for the particular class ofthe first antibody. For example, if an anti-caspase-14 antibody is ofthe IgG class, a second antibody will be an anti-IgG antibody. Suchsecond antibodies are readily available from commercial sources. Thesecond antibody can be labeled using a detectable moiety as describedabove. When a sample is labeled using a second antibody, the sample isfirst contacted with a first antibody, which is an anti-caspase-14antibody, then the sample is contacted with the labeled second antibody,which specifically binds to the anti-caspase-14 antibody and results ina labeled sample.

Methods for raising polyclonal antibodies, for example, in a rabbit,goat, mouse or other mammal, are well known in the art. In addition,monoclonal antibodies can be obtained using methods that are well knownand routine in the art (Harlow and Lane, supra, 1992). For example,spleen cells from a caspase-14-immunized mammal can be fused to anappropriate myeloma cell line such as SP/02 myeloma cells to producehybridoma cells. Cloned hybridoma cell lines can be screened using alabeled caspase-14 polypeptide or functional fragment thereof toidentify clones that secrete anti-caspase-14 monoclonal antibodieshaving the desired specificity. Hybridomas expressing anti-caspase-14monoclonal antibodies having a desirable specificity and affinity can beisolated and utilized as a continuous source of the antibodies, whichare useful, for example, for preparing standardized kits as describedabove. Similarly, a recombinant phage that expresses, for example, asingle chain anti-caspase-14 also provides a monoclonal antibody thatcan used for preparing standardized kits.

A monoclonal anti-caspase-14 antibody can be used to prepareanti-idiotypic antibodies, which present an epitope that mimics acaspase-14-specific epitope recognized by the monoclonal antibody usedto prepare the anti-idiotypic antibodies. Where the epitope mimickedincludes, for example, a portion of the caspase-14 catalytic domain, theanti-idiotypic antibody can act as a competitor of caspase-14 and,therefore, can be useful for reducing the level of activity ofcaspase-14 and, consequently, the level of apoptotic activity. Thus, theinvention further provides an anti-idiotypic anti-caspase-14 antibody,which mimics a caspase-14-specific epitope, such as an epitope of SEQ IDNOS:2, 5, 7, or 9, an epitope of the large or small subunit of acaspase-14 polypeptide or an epitope of a caspase-14 heterodimer orheterotetramer.

The invention also provides an isolated nucleic acid molecule encoding acaspase-14 polypeptide or functional fragment thereof. The term“isolated” means in a form that is relatively free from contaminatinglipids, polypeptides, unrelated nucleic acid molecules and othercellular material normally associated with the nucleic acid molecule inthe cell and at least 30% of the total material. In other embodiments ofthe invention, the nucleic acid molecule is 50% or 70% of the totalmaterial. In other embodiments of the invention, the nucleic acidmolecule is 90% or 95% of the total material. In yet another embodimentof the invention, the nucleic acid molecule is greater than 95% of thetotal material. Thus, an isolated nucleic acid molecule of the inventionis one that is in a form that is different from the naturally occurringstate.

One exemplary nucleic acid molecule of the invention is provided by SEQID NO:1, which is 850 nucleotides in length and encodes SEQ ID NO:2 (seeFIG. 1). Another exemplary nucleic acid molecule is provided by SEQ IDNO:4, which is 777 nucleotides in length and encodes SEQ ID NO:5 (seeFIG. 7). Additional nucleic acid molecules of the invention are thosethat have an oligonucleotide or polynucleotide sequence that encodes SEQID NOS:2 or 5 or a functional fragment thereof. In addition, theinvention provides nucleic acid molecules that have an oligonucleotideor polynucleotide sequence that encodes a caspase-14 polypeptide or afunctional fragment thereof such as SEQ ID NOS:6 and 8.

Such an oligonucleotide or polynucleotide sequence also can be useful,for example, as a probe or a PCR primer. Such probes can be used toscreen a genomic DNA library or a cDNA library to obtain other nucleicacid molecules encoding caspase-14 polypeptides or to diagnose a diseaseassociated with enhanced or inhibited apoptosis (see below). Thus, theinvention provides oligonucleotide sequences that comprise at least 12contiguous nucleotides of SEQ ID NOS:1, 4, 6, or 8. In otherembodiments, the invention provides oligonucleotide sequences thatcomprise at least 15, 18 or 21 contiguous nucleotides of SEQ ID NOS:1,4, 6, or 8. In another embodiment, the invention provides a nucleic acidmolecule encoding SEQ ID NOS:2, 5, or splice variants thereof. In yetanother embodiment, the invention provides a nucleic acid moleculeencoding a caspase-14 polypeptide. Oligonucleotide sequences consistingof nucleotide positions 454 to 474 or positions 460 to 477 of SEQ IDNO:1, or nucleotide positions 430 to 450 or positions 436 to 453 of SEQID NO:4 and/or homologous positions of splice variant forms, or anycontiguous portion thereof, however, are not encompassed within thenucleic acid molecules of the invention. Similarly, nucleic acidmolecules that consist of the expressed sequence tag having GenBankaccession number AA103647, or any contiguous portion thereof, also arenot encompassed within the nucleic acid molecules of the invention.

In another embodiment, the invention provides an isolated gene encodingcaspase-14, as well as functional fragments of a caspase-14 gene. A geneencoding caspase-14 can be obtained by screening a genomic libraryusing, for example, an oligonucleotide or polynucleotide sequence of SEQID NOS:1, 4, 6, or 8 as a probe, as discussed above. Methods ofpreparing genomic libraries are known in the art (Perbal, Bernard, APractical Guide to Molecular Cloning, John Wiley & Sons, Inc. (1988),ch. 17, and the various references cited therein).

In addition, nucleic acid molecules that do not encode a caspase-14 geneproduct but, instead, are regulatory elements are considered part of thegene encoding caspase-14, particularly functional fragments of acaspase-14 gene. Specific examples of such functional fragments of acaspase-14 gene include promoters, enhancers and other gene expressionregulatory elements present in a caspase-14 encoding gene. Thus, uponobtaining a caspase-14 gene, as described above, regulatory elementspresent in the caspase-14 gene can be identified using routine methods.

To identify sequences having homology to the caspase family ofproteases, nucleic acid molecules encoding apoptotic cysteine proteasescan be enriched by PCR amplification of a cDNA library using a primerdesigned to encompass homologous regions in nucleic acid sequences thatencode known caspase protease family members. The enriched library canbe further amplified by PCR using a primer with sequences havinghomology to the putative novel protease cDNA but not to the othercaspase family of proteases. For example, to obtain a caspase-14polypeptide, such as a mammalian homologue of SEQ ID NOS:2, 5, 7, or 9,a primer with sequences homologous to SEQ ID NOS:1, 4, 6, or 8,respectively, but not to the other caspases can be used.

As searching a genetic data base will yield homologous sequence matchesto any query nucleotide sequence, additional criteria must be used toidentify authentic caspase homologs from non-specific matches. Caspasefamily members share the highest degree of homology in the active siteand catalytically important amino acid residues (FIG. 2). A given ESTreturned by a search may not necessarily include one of these highlyhomologous sites but, rather, may only include a region within theprotease having cryptic homology. Confirming an EST as encoding part ofa novel caspase protease involves translation of all the positive ESThits in three different reading frames and subsequent identification ofconservative active site or catalytically important amino acid sequencemotifs. Then, using conventional cDNA cloning, a full length cDNA of theputative novel protease can be obtained and 1) analyzed for overallstructural homology to caspase family members, 2) recombinantlyexpressed and analyzed for cysteine protease activity, and 3) analyzedfor the induction of apoptosis by heterologous expression of the cDNA inappropriate cells.

Alternative methods for isolating a caspase-14 encoding nucleic acidmolecule also can be employed. For example, using the teachingsdescribed herein, those skilled in the art can routinely isolate andmanipulate caspase-14 nucleic acid molecules using well known methods.All that is necessary is a disclosed sequence of a caspase-14 encodingnucleic acid molecule, for example, SEQ ID NOS:1, 4, 6, or 8, or itsdeduced amino acid sequence, for example, SEQ ID NOS:2, 5, 7, or 9,respectively. Such methods include, for example, screening a cDNA orgenomic library by using synthetic oligonucleotides, nucleic acidfragments or primers as hybridization probes. Alternatively, antibodiesto a caspase-14 polypeptide or functional fragment thereof, particularlyto a caspase-14-specific epitope, can be generated and used to screen anexpression library to isolate caspase-14 encoding nucleic acids.

The above described methods are known to those skilled in the art(Sambrook et al., supra, 1989, and the various references cited therein;Ansubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md., Supp. 39 (1997)). Furthermore, recombinant DNAmethods currently used by those skilled in the art include thepolymerase chain reaction (PCR), which, combined with the caspase-14nucleotide and amino acid sequences described herein, allowsreproduction of caspase-14 encoding sequences. Desired sequences can beamplified exponentially starting from as little as a single gene copy bymeans of PCR. The PCR technology is the subject matter of U.S. Pat. Nos.4,683,195, 4,800,159, 4,754,065, and 4,683,202.

A caspase-14 nucleic acid molecule of the invention, as well as acaspase-14 polypeptide or functional fragment thereof, can be used todiagnose, or to generate reagents to diagnose, pathological conditionsassociated with increased or decreased levels of apoptosis. Such methodsof diagnosis include using a nucleic acid probe, which can hybridizewith a caspase-14 containing nucleotide sequence, or using an antibodyor ligand, which binds a caspase-14 polypeptide. Methods of diagnosisfurther include detecting caspase-14 enzymatic activity in a sample (seeExample 2). Such methods, which are disclosed herein or otherwise knownin the art, can be performed ex vivo, for example, by removing a cell ortissue sample from an individual exhibiting or suspected of having apathological condition associated with increased or decreased levels ofapoptosis. Correlation of increased caspase-14 expression or activity,as compared to normal levels of caspase-14, which can be determined bytaking samples from apparently normal individuals, is indicative of adisease associated with increased levels of apoptosis, whereascorrelation of decreased caspase-14 expression or activity is indicativeof a disease associated with decreased levels of apoptosis. As usedherein, reference to “increased” or “decreased” expression or activityof caspase-14 or “increased” or “decreased” levels of apoptosis means atleast about one standard deviation and, preferably, at least about twostandard deviations, above or below, respectively, the normal expressionor activity or levels of caspase-14 in a corresponding sample of anormal individual.

A caspase-14 encoding nucleic acid of the invention, as well as acaspase-14 polypeptide or functional fragment thereof, can be used toreduce the severity of a pathological condition characterized, in part,by increased or decreased levels of apoptosis. A caspase-14 polypeptideor functional fragment thereof that includes, for example, the catalyticdomain of caspase-14 can be formulated into a pharmaceutical compositionand, therefore, can be used as a medicament. Such a medicament is usefulin the treatment of an individual having a disease characterized, inpart, by decreased levels of apoptosis, which is associated withincreased cell survival and proliferation. Such a caspase-14 polypeptideor functional fragment thereof can increase the levels of apoptosis inan individual with such a disease and, thereby, decrease cell survivaland proliferation. Examples of pathological conditions associated withdecreased levels of apoptosis and, therefore, increased cell survivalinclude cancers such as lymphomas and hormone dependent tumors such asbreast, prostate and ovarian cancer, autoimmune diseases such assystemic lupus erythematosus, immune-mediated glomerulonephritis andviral infections such as herpesvirus, poxvirus and adenovirus.

Additionally, molecules that interact with caspase-14, directly orindirectly, to induce caspase-14 mediated apoptosis can be used to treatsuch a disease. Such molecules that interact directly with caspase-14can be identified based on their physical association with caspase-14using, for example, an affinity matrix comprising caspase-14 or a methodsuch as the two hybrid assay (U.S. Pat. No. 5,283,173).

To be effective, caspase-14 polypeptides or functional fragments thereofmust be introduced into cells characterized by decreased levels ofapoptosis. Introduction can be accomplished by a variety of means knownin the art including, for example, using lipid vesicles or receptormediated endocytosis. Targeting the appropriate cell type also can beaccomplished by conjugating the caspase-14 polypeptide or functionalfragment thereof to a specific receptor ligand or a target cell specificantibody, producing a caspase-14 fusion protein comprising the ligand orantibody.

In contrast to the induction of caspase-14 mediated apoptosis for thetreatment of pathological conditions characterized by increased cellsurvival or proliferation, inhibitors of caspase-14 can be used to treatpathological conditions associated with increased levels of apoptosis.Examples of pathological conditions associated with increased levels ofapoptosis and, therefore, decreased cell survival include, for example,degenerative disorders such as Alzheimer's disease, Parkinson's disease,amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellardegeneration, myelodysplastic syndromes such as aplastic anemia andischemic injury, including myocardial infarction, stroke and reperfusioninjury.

Such inhibitors of caspase-14 can be, for example, inhibitors of thecaspase-14 protease activity or inhibitors of the conversion of theinactive, proenzyme into the active caspase-14 protease. Specificexamples of such inhibitors can include, for example, anti-caspase-14antibodies, proteins, small peptidyl protease inhibitors and smallnon-peptide, organic molecule inhibitors. Such inhibitors are formulatedin a medium that allows introduction into the desired cell type.Alternatively, such inhibitors can be attached to targeting ligands forintroduction by cell mediated endocytosis and other receptor mediatedevents. Specific caspase-14 peptidyl inhibitors can include suicideinhibitors and substrate analogues such as the tetrapeptide DEVDaldehyde and the cowpox virus protein Crm A, for example.

Other inhibitors of caspase-14 include, for example, small molecules ororganic compounds that bind and inactivate caspase-14 by a competitiveor non-competitive inhibitory type mechanism. Molecules or compoundsthat indirectly inhibit the caspase-14 pathway can also be used asinhibitors of caspase-14. Caspase-14 inhibitors can be identified byscreening for molecules that demonstrate specific or beneficialcaspase-14 inhibitory activity. Such methods are described herein andcan be practiced by those skilled in the art in view of the disclosedcaspase-14 nucleotide sequences and amino acid sequences.

Dominant/negative inhibitors of caspase-14 also can be used to treat orreduce the severity of pathological conditions associated with enhancedapoptosis. For example, a dominant/negative inhibitor comprising thelarge subunit of caspase-14, but lacking the active site QACRG (SEQ IDNO:3; positions 134-138 of SEQ ID NO:2; positions 130-134 of SEQ ID NO:5or 7; positions 102-106 SEQ ID NO:9), can bind the small subunit ofcaspase-14 to form a complex that lacks protease activity. Such amechanism of dominant negative inhibition of caspase-14 is similar tothe dominant negative inhibition due to alternately spliced isoforms ofcaspase-2, caspase-7 and caspase-8 (Cohen, J. Biochem. 326:1-16 (1997)).Subunits from other caspases similarly can be used to formdominant/negative inhibitors of caspase-14 activity and, therefore, totreat pathological conditions associated with increased levels ofapoptosis. Such subunits should be selected so that they bind either thelarge or small subunit of caspase-14 polypeptides to prevent theirassembly into active heterotetrameric protease complexes. Ananti-idiotypic anti-caspase-14 antibody also can serve this purpose.Moreover, caspase-14 subunits that have been modified so as to becatalytically inactive can be used as dominant/negative inhibitors ofcaspase-14. Such modifications include, for example, mutation of theactive site cysteine residue (amino acid position 136 of SEQ ID NO:2;amino acid position 132 of SEQ ID NO:5) to another amino acid such asalanine or glycine.

Caspase-14 substrate antagonists also can be used to treat or reduce theseverity of pathological conditions associated with increased levels ofapoptosis. Such substrate antagonists can bind to and inhibit cleavageby caspase-14, thereby preventing commitment progression of apoptosisSubstrate antagonists include, for example, ligands and small moleculecompounds.

A caspase-14 polypeptide or functional fragment thereof, or an inhibitorof caspase-14, can be administered by conventional therapeutic methods,in dosages that are sufficient to respectively increase or decrease thelevels of apoptosis in the target cells. Such dosages can be determinedby those skilled in the art using, for example, Phase I and Phase IItrials. Administration can be accomplished by injection, for example,intravenous, intraperitoneal or subcutaneous injection, and can beperformed in a variety of different regimes, including single high doseadministration, repeated small dose administration or a combination ofboth. The dosing will depend on the cell type, progression of thepathological condition and the overall health of the individual.

Treatment or reduction of the severity of pathological conditionsassociated with increased or decreased levels of apoptosis also can beaccomplished by introducing expressible nucleic acid molecules encodingrespectively caspase-14 polypeptides or functional fragments thereof orcaspase-14 inhibitors such as antisense caspase-14 nucleic acidmolecules into cells characterized by such pathological conditions. Forexample, treatment to reduce the severity of a pathological conditionassociated with decreased levels of apoptosis can be accomplished byelevating the synthesis rates of caspase-14 using recombinant caspase-14expression vectors and gene transfer technology. Conversely, treatmentor reduction of the severity of pathological conditions associated withincreased levels of apoptosis can be accomplished by introducing andexpressing antisense caspase-14 nucleic acid molecules, which inhibitendogenous caspase-14 expression. Such methods of introduction andexpression are well known in the art and described below with referenceto recombinant viral vectors. Other vectors compatible with theappropriate targeted cell can accomplish the same goal and can besubstituted in the methods described herein in place of recombinantviral vectors.

Further embodiments include the inhibition of neoplasia or apoptosis byutilizing specific antisense polynucleotides complementary to all orpart of the nucleic acid sequences SEQ ID NOS:1, 4, 6, or 8 encoding acaspase-14. Such complementary antisense polynucleotides may includesubstitutions, additions, deletions, or transpositions, as long asspecific hybridization to the relevant target sequence in SEQ ID NOS:1,4, 6, or 8 is retained as a functional property of the polynucleotide.Antisense polynucleotides that prevent transcription and/or translationof mRNA corresponding to caspase-14 may inhibit apoptosis. Antisensepolynucleotides of various lengths may be produced and used, however,the sequence length is typically at least 20 consecutive nucleotidesthat are substantially or wholly identical to the sequence of SEQ IDNOS:1, 3, 4, 6, or 8. (see U.S. Pat. No. 5,691,179 and Antisense RNA andDNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. 1988, each of which is incorporated herein by reference).

Recombinant viral vectors are useful for in vivo expression of a desirednucleic acid molecule because such vectors can offer advantages such aslateral infection and targeting specificity. Lateral infection isinherent in the life cycle of viral vectors such as retroviruses and isthe process by which a single infected cell produces many progenyvirions that bud off and infect neighboring cells. Lateral infection canresult in rapid infection of a large area, most of which were notinitially infected by the original viral particles. Viral vectors thatare unable to spread laterally can be useful where it is not necessaryto introduce a specified gene into all of the targeted cells.

Typically, viruses infect and propagate in specific cell types.Therefore, viral vectors are useful for specifically introducing adesired gene into predetermined cell types. The vector to be used in themethods of the invention will depend on desired cell type to betargeted. For example, if neurodegenerative diseases are to be treatedby decreasing the caspase-14 activity of affected neuronal cells, then avector specific for cells of the neuronal cell lineage, for example,herpesvirus based vectors, should be used (Kaplitt and Loewy, ViralVectors, Academic Press, Inc. (1995)). Similarly, if diseases orpathological conditions of the hematopoietic system are to be treated,then a viral vector such as an HIV based vector that is specific forblood cells and their precursors, preferably for a specific type ofhematopoietic cell, should be used. Moreover, such vectors can bemodified with specific receptors or ligands to modify target specificitythrough receptor mediated events. These modification procedures can beperformed by recombinant DNA techniques or synthetic chemistryprocedures or the like. The specific type of vector will depend upon theintended application. Thus, the invention provides a vector thatcontains a nucleic acid molecule encoding a caspase-14 polypeptide orfunctional fragment thereof. As described herein, vectors of theinvention can be used in an appropriate host cell. Thus, the inventionprovides a cell containing a vector of the invention. Vectors of theinvention are known and readily available within the art or can beconstructed by one skilled in the art using well known methodology.

A vector of the invention, such as one encoding a caspase-14 polypeptideor an inhibitor of caspase-14, for example, an antisense nucleic acidmolecule, can be administered in several ways to obtain expression ofsuch a sequence, which can increase or decrease, respectively, the levelof activity of caspase-14 in the cells affected by the disease orpathological condition. If a viral vector is used, the procedure cantake advantage of their target specificity and, consequently, a vectordoes not necessarily have to be administered locally at the diseasedsite. However, local administration can provide a quicker and moreeffective treatment. Administration can be performed by conventionalmethods, for example, intravenous or subcutaneous injection into thesubject. Injection of a viral vector into the spinal fluid also can beused as a mode of administration, especially in the case ofneurodegenerative diseases of the central nervous system. Followinginjection, the viral vector will bind to a target cell expressing anappropriate receptor.

A caspase-14 encoding vector can be administered locally at the site ofthe disease or pathological condition. Local administration isadvantageous because there is no dilution effect and, therefore, asmaller dose is required to achieve caspase-14 expression in a majorityof the targeted cells. Additionally, local administration can alleviatethe targeting requirement of other forms of administration, since avector can be used that infects all cells in the locally administeredarea. If expression is desired in only a specific subset of cells withinthe administered area, then promoter and expression elements that arespecific for the desired subset can be incorporated in the vector, whichcan be a viral vector, viral genome, plasmid or phagemid. A transfectionvehicle such as a liposome can be used to introduce the vector intorecipient cells within the inoculated area. Such transfection vehiclesare known by those skilled in the art. Alternatively, the vector can beadministered directly into a tissue of an individual (Wolff et al.,Science 247:1465-1468 (1990)).

Additional features can be added to a vector to ensure safety and/orenhance therapeutic efficacy. Such features include for example, markersthat can be used to negatively select against cells infected with therecombinant virus. An example of such a negative selection marker is theTK gene that confers sensitivity to the antibiotic gancyclovir. Negativeselection is a means by which infection can be controlled because itprovides inducible suicide through the addition of antibiotic.

Additionally, a caspase-14 encoding nucleic acid molecule of theinvention, as well as a caspase-14 polypeptide or functional fragmentthereof, can be used to screen for pharmaceutical compounds andmacromolecules that modulate, that is, inhibit or enhance, caspase-14activity. Such a caspase-14 encoding nucleic acid molecule, caspase-14polypeptide or functional fragment thereof can be used in a sample toscreen for inhibitors of caspase-14, including those that inhibitenzymatic or apoptotic activity. Alternatively, a caspase-14 encodingnucleic acid molecule, caspase-14 polypeptide or functional fragmentthereof can be used in a sample to screen for compounds that enhancecaspase-14 activity such as by inducing cleavage of the caspase-14proenzyme into its active subunits. Such a sample can contain a celllysate and also can contain isolated caspase-14 encoding nucleic acidmolecules, caspase-14 polypeptides or functional fragments thereof.

Candidate inhibitors and enhancers may be isolated or procured from avariety of sources, such as bacteria, fungi, plants, parasites,libraries of chemicals, peptides or peptide derivatives and the like.Inhibitors and enhancers may be also be rationally designed, based onthe protein structure determined from X-ray crystallography (see, Mittlet al., J. Biol. Chem., 272:6539-6547, 1997). In certain preferredembodiments, the inhibitor targets a specific caspase (e.g., caspase-3and not any other caspases).

Without being held to a particular mechanism, the inhibitor may act bypreventing processing of caspase or by preventing enzymatic activity, orby other mechanism. The inhibitor may act directly or indirectly. Inpreferred embodiments, inhibitors interfere in the processing of thecaspase protein. In other preferred embodiments, the inhibitors aresmall molecules. In a most preferred embodiment, the inhibitors preventapoptosis. Inhibitors should have a minimum of side effects and arepreferably non-toxic. Inhibitors that can penetrate cells are preferred.

In addition, enhancers of caspase activity or expression are desirablein certain circumstances. At times, increasing apoptosis will have atherapeutic effect. For example, tumors or cells that mediate autoimmunediseases are appropriate cells for destruction. Enhancers may increasethe rate or efficiency of caspase processing, increase transcription ortranslation, or act through other mechanisms. As is apparent to oneskilled in the art, many of the guidelines presented above apply to thedesign of enhancers as well.

Screening assays for inhibitors and enhancers will vary according to thetype of inhibitor or enhancer and the nature of the activity that isbeing affected. Assays may be performed in vitro or in vivo. In general,in vitro assays are designed to evaluate caspase protein processing orcaspase enzymatic activity, and in vivo assays are designed to evaluatecaspase protein processing, caspase enzymatic activity, apoptosis, orcaspase cleavage of substrate. In any of the assays, a statisticallysignificant increase or decrease compared to a proper control isindicative of enhancement or inhibition.

One type of in vitro assay can be performed by examining the effect of acandidate compound on the processing of caspase-14 into two subunits.Briefly, a caspase-14, that is a primary translation product, isobtained from an in vitro translation system. The caspase-14 ispreferably constructed to be capable of normal auto-processing, but canbe constructed to be cleaved by other protease components present oradded to the reaction. This primary product is contacted with orwithout, or translated in the presence or absence of a candidatecompound and assessed for appearance of the two subunits. To facilitatedetection, typically, the caspase-14 is labeled during translation. Thetwo subunits may be readily detected by autoradiography after gelelectrophoresis. One skilled in the art will recognize that othermethods of labeling and detection may be used alternatively.

An alternative in vitro assay is designed to measure cleavage of acaspase substrate (e.g., Acetyl DEVD-aminomethyl coumarin (amc), lamin,PRPP, and the like). Substrate turnover may be assayed using eithercleavable or noncleavable rev-caspase. Briefly, in this method,caspase-14 is translated and allowed sufficient time to be processed orsubjected to a protease which activates caspase-14. The caspasesubstrate along with the candidate compound is added to the reaction.Detection of cleaved substrate is performed by any one of a variety ofstandard methods. Generally, the substrate will be labeled and followedby an appropriate detection means.

Moreover, any known enzymatic analysis can be used to follow theinhibitory or enhancing ability of a candidate compound with regard to acaspase-14 of this invention. For example, one could express caspase-14in a cell line be it bacterial, insect, mammalian or other, and purifythe caspase. The purified caspase-14 could then be used in a variety ofassays to follow its catalytic ability in the presence of candidatecompounds, as noted above. Such methods of expressing and purifyingrecombinant proteins are known in the art and examples can be found inSambrook et al., Molecular Cloning: A Laboratory Manual Cold SpringHarbor Press, 1989 as well as in a number of other sources.

In vivo assays are typically performed in cells transfected eithertransiently or stably with an expression vector containing a caspase-14gene, such as those described herein. These cells are used to measurecaspase-14 processing, substrate turnover, or apoptosis in the presenceor absence of a candidate compound. When assaying apoptosis, a varietyof cell analyses may be used including, for example, dye staining andmicroscopy to examine nucleic acid fragmentation and porosity of thecells. Further, in vivo assaying for the ability of the transfectedcaspase-14 to cleave known substrates that are co-transfected or placedin the cell culture media in the presence of the candidate compound canbe performed thereby allowing for the detection and determination ofsubstrate turnover.

The assays briefly described herein may be used to identify an enhanceor inhibitor that is specific for an individual caspase. In a preferredembodiment candidate compounds would be analyzed using a variety ofcaspases (e.g., caspase-1 through caspase-14) to identify specificinhibitors and enhancers for individual caspases.

A variety of methodologies exist can be used to investigate the effectof a candidate compound Such methodologies are those commonly used toanalyze enzymatic reactions and include, for example, SDS-PAGE,spectroscopy. HPLC analysis, autoradiography, chemiluminescence,chromogenic reactions, and immunochemistry (e.g., blotting,precipitating, etc.).

Inhibitors and enhancers may be used in the context of this invention toexert control over the cell death process or cytokine activation. Thus,these inhibitors and enhancers will have utility in diseasescharacterized by either excessive or insufficient levels of apoptosis.Inhibitors of caspase proteases have potential to treat the majorneurodegenerative diseases: stroke, Parkinson's Disease, Alzheimer'sDisease, and ALS. As well, caspase-14 protease inhibitors may be used toinhibit apoptosis in the heart following myocardial infarction, in thekidney following acute ischemia, and in diseases of the liver. Enhancersof caspase-14 activity may be used in contexts when apoptosis orcytokine activation are desired. For example, inducing or increasingapoptosis in cancer cells or aberrantly proliferating cells may beeffected by delivery of a caspase enhancer.

Such screening methods are known to those skilled in the art and can beperformed by either in vitro or in vivo procedures. For example, Example2 provides a specific in vitro assay for caspase-14 protease activity.This assay employs a sample containing a caspase-14 polypeptideexpressed in an active, processed form recombinantly in E. coli. Theprotease activity of the polypeptide is measured by incubation with afluorescent substrate. This assay can be used to screen synthetic ornaturally occurring compound libraries, including macromolecules, foragents that either inhibit or enhance caspase-14 activity. Thecaspase-14 polypeptides or functional fragments thereof to be used inthe assay can be obtained by, for example, in vitro translation,recombinant expression or biochemical procedures. Methods other thanthat described in Example 2 also can be used to screen and identifycompounds that inhibit or enhance caspase-14 activity including, forexample, those described supra and other methodologies such as usingphage display peptide libraries, where greater than 108 peptidesequences can be screened in a single round of panning. Such methods, aswell as others, are known in the art and can be utilized to identifycompounds that inhibit or enhance caspase-14 activity.

As noted above, caspase-14 nucleic acid molecules may be delivered tocells in combination with a vector or other gene delivery vehicle. Thesemethods may be accomplished by delivery of DNA or cDNA capable of invivo transcription caspase-14 or an active fragment thereof. Morespecifically, in order to produce caspase-14 in vivo, a nucleic acidsequence coding for caspase-14 is placed under the control of aeukaryotic promoter (e.g., a pol III promoter, CMV or SV40 promoter).Where it is desired to more specifically control transcription, thecaspase-14 encoding nucleic acid molecule may be placed under thecontrol of a tissue or cell specific promoter (e.g., to target cells inthe liver), or an inducible promoter, such as MMTV LTR, CMV IE promoter,RSV LTR, SV40, metallothionein promoter (see, e.g., U.S. Pat. No.4,870,009), ecdysone response element system, tetracycline-reversiblesilencing system (tet-on, tet-off), and the like.

Many techniques for introduction of nucleic acids into cells are known.Such methods include retroviral vectors and subsequent retrovirusinfection, adenoviral or adeno-associated viral vectors and subsequentinfection, and complexes of nucleic acid with a condensing agent (e.g.,poly-lysine). These complexes or viral vectors may be targeted toparticular cell types by way of a ligand incorporated into the vehicle.Many ligands specific for tumor cells and other cells are well known inthe art.

A wide variety of vectors may be utilized within the context of thepresent invention, including for example, plasmids, viruses,retrotransposons and cosmids. Representative examples include adenoviralvectors (e.g., WO 94/26914, WO 93/9191; Yei et al., Gene Therapy1:192-200, 1994; Kolls et al., PNAS 91(1):215-219, 1994; Kass-Eisler etal., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation88(6):2838-48, 1993; Guzman et ale, Cir. Res. 73(6):1202-1207, 1993;Zabner et al., Cell 75(2):207-216, 1993; Li et al., Hum Gene Ther.4(4):403-409, 1993; Caillaud et al., Eur. J. Neurosci. 5(10):1287-1291,1993), adeno-associated type 1 (“AAV-1”) or adeno-associated type 2(“AAV-2”) vectors (see WO 95/13365; Flotte et al., PNAS90(22):10613-10617, 1993), hepatitis delta vectors, live, attenuateddelta viruses and herpes viral vectors (e.g., U.S. Pat. No. 5,288,641),as well as vectors which are disclosed within U.S. Pat. No. 5,166,320.Other representative vectors include retroviral vectors (e.g., EP 0 415731: WO 90/07936; WO 91/02805; WO 94/03622; WO 93/25698; WO 93/25234;U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218.

Within certain aspects of the invention, nucleic acid molecules thatencode caspase-14 may be introduced into a host cell utilizing a genedelivery vehicle, or by various physical methods. Representativeexamples of such methods include transformation using calcium phosphateprecipitation (Dubensky et al., PNAS 81:7529-7533, 1984), directmicroinjection of such nucleic acid molecules into intact target cells(Acsadi et al., Nature 352:815-818, 1991), and electroporation wherebycells suspended in a conducting solution are subjected to an intenseelectric field in order to transiently polarize the membrane, allowingentry of the nucleic acid molecules. Other procedures include the use ofnucleic acid molecules linked to an inactive adenovirus (Cotton et al.,PNAS 89:6094, 1990), lipofection (Felgner et al., Proc. Natl. Acad. Sci.USA 84:7413-7417, 1989), microprojectile bombardment (Williams et al.,PNAS 88:2726-2730, 1991), polycation compounds such as polylysine,receptor specific ligands, liposomes entrapping the nucleic acidmolecules, spheroplast fusion whereby E. coli containing the nucleicacid molecules are stripped of their outer cell walls and fused toanimal cells using polyethylene glycol, viral transduction, (Cline etal., Pharmac. Ther. 29:69, 1985; and Friedmann et al., Science 244:1275,1989), and DNA ligand (Wu et al., J. Biol. Chem. 264:16985-16987, 1989),as well as psoralen inactivated viruses such as Sendai or Adenovirus. Inone embodiment, the caspase-14 encoding construct is introduced into thehost cell using a liposome.

In an additional embodiment, the compositions of caspase-14 may beadministered either alone, or as a pharmaceutical composition. Thesecompositions may contain any of the above described inhibitors,enhancers, DNA molecules, vectors or host cells, along with apharmaceutically or physiologically acceptable carrier, excipients ordiluents. Generally, such carriers should be nontoxic to recipients atthe dosages and concentrations employed. Ordinarily, the preparation ofsuch compositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents.

Compositions of the present invention may be formulated for the mannerof administration indicated, including for example, for oral, nasal,venous, intracranial, intraperitoneal, subcutaneous, or intramuscularadministration. Within other embodiments of the invention, thecompositions described herein may be administered as part of a sustainedrelease implant. Within yet other embodiments, compositions of thepresent invention may be formulized as a lyophilizate, utilizingappropriate excipients which provide stability as a lyophilizate, andsubsequent to rehydration. One skilled in the art may further formulatethe enhancers or inhibitors of this invention in an appropriate manner,and in accordance with accepted practices, such as those disclosed inRemington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co.,Easton, Pa. 1990. Pharmaceutical compositions are useful for bothdiagnostic or therapeutic purposes.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease. Dosages may be determined most accurately duringclinical trials. Patients may be monitored for therapeutic effectivenessby appropriate technology, including signs of clinical exacerbation,imaging and the like.

It is understood that modifications that do not substantially affect thevarious embodiments of the invention also are included within theinvention. Accordingly, the following examples are intended toillustrate but not limit the present invention.

EXAMPLES Example 1 Characterization of Caspase-14

This example shows the sequencing and analysis of caspase-14.

An EST, GenBank accession number AA103647, was identified during ahomology search of the GenBank database using a query nucleotidesequence based on caspase-3 and caspase-6 coding sequences. The EST is asequence of 483 nucleotides in length.

The EST was derived from a mouse cell clone, which was obtained fromIMAGE Consortium. The EST was resequenced, revealing that it containednumerous sequencing errors, including at nucleotide positions 13, 54 and164 of SEQ ID NO:1, where the corresponding positions in the EST containnothing. Full sequencing of the clone revealed a sequence of 850nucleotides in length encoding a polypeptide (SEQ ID NO:2), designatedherein as caspase-14, which is similar to members of the caspase familyof proteases.

Following this characterization, amplification of a partial humancaspase-14 cDNA from a human brain cDNA library was conducted. The setof forward and reverse primers derived from the mouse cDNA sequence ofcaspase-14 are set forth below:

(SEQ ID NO: 69) Forward primer: ATATGATATGTCAGGTGCCCG (SEQ ID NO: 70)Reverse primer: TTCCGGAGGGTGCTTTGGA

To obtain the 5′ and 3′ coding sequences of human caspase-14 weperformed RACE (rapid amplification of cDNA ends) using nested PCRprimers derived from the human caspase-14 cDNA and vector specificprimers complimentary to the library vector.

For 5′ amplification Reverse primers: CCTGTATGATGTACACCTTGG (SEQ ID NO:71) AGAGATTCTCCAGCTTGAC (SEQ ID NO: 72) ATCTTCTCCCTTGAGGAAG (SEQ ID NO:73) For 3′ amplification Forward primers: ATATGATATGTCAGGTGCCCG (SEQ IDNO: 74) CAAGGTGTACATCATACAGG (SEQ ID NO: 75)

Following the above PCR amplification, the derived sequence (SEQ IDNO:4) and the predicted amino acid sequence (SEQ ID NO:5) were comparedwith the mouse sequence. The human and mouse proteins exhibit 75%identity when compared using the GCG pileUp program (Gapweight: 12,Gaplength weight: 4). The pileUp program creates a multiple sequencealignment form a group of related sequences using progressive, pairwisealignments. PileUp creates a multiple sequence alignment using theprogressive alignment method of Feng and Doolittle (J. Mol. Evol.25:351-360, 1987) and is similar to the method described by Higgins andSharp (CABIOS 5:151-153, 1989).

Example 2 Kinetic Parameters of Caspase-14

This example provides methods to characterize the protease activity andsubstrate specificity of caspase-14.

The kinetic properties of bacterially expressed recombinant caspase-14is determined using tetrapeptide substrates in a continuous fluorometricassay. Examples of two such substrates are DEVD-AMC and the YVAD-AMC,which represent the cleavage sites for the poly(ADP-ribose)polymerase(PARP) and IL-1β P1-P4 substrate tetrapeptides, respectively (Nicholsonet al., Nature 376:37-43 (1995)). Caspase-14 cDNA lacking most of thepropeptide coding sequence is subcloned in-frame into the Bam HI/XhoIsites of the bacterial expression vector pGEX-5X-3 (Pharmacia BiotechInc.). This vector produces caspase-14 as a fusion protein withglutathione S-transferase (GST) and is used essentially as described inFernandes-Alnemri et al., Cancer Res. 55:6045-6052 (1995). TheGST-caspase-14 expression vector is constructed and transformed intoDH5α bacteria using routine molecular biology methods known to thoseskilled in the art. After induction with IPTG, bacterial extracts areprepared from E. coli expressing the recombinant fusion proteins. Theextracts are adsorbed to glutathione-Sepharose resin, washed severaltimes and then analyzed by SDS-PAGE.

The isolated caspase-14 GST-fusion protein is then used for furtherenzymatic analyses. The activity of caspase-14 is measured usingbacterial lysates prepared with ICE buffer (25 mM HEPES, 1 mM EDTA, 5 mMDTT, 0.1% CHAPS, 10% sucrose, pH 7.5) at room temperature (24-25° C.).The K_(i)'s are determined from the hydrolysis rate of 50 μM DEVD-AMCfollowing a 30 min preincubation of the enzyme with inhibitors DEVD-CHOand recombinant CrmA protein. Prior to incubation with enzyme, purifiedCrmA is activated by incubation with 5 mM DTT for 10 min at 37° C.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention.

Example 3 Overexpression of Procaspase-14 in MCF-7 Cells

Overexpression of small prodomain executioner procaspases such asprocaspase-3 or -6 in mammalian cells does not induce apoptosis, due totheir inability to autoprocess themselves. On the other handoverexpression of the large prodomain initiator procaspases such asprocaspase-8 can induce apoptosis, due to their ability to undergoprodomain-mediated oligomerization. To test the ability of procaspase-14to induce apoptosis in transfected cells, MCF-7 cells were transientlytransfected with procaspase-14 in a PRSC lac-Z expression constructunder the CMV promoter. Cells were also transfected with an empty vectoror constructs encoding procaspase-3, -6, -8, or -10 as controls. Thecells were stained with X-gal 30 h after transfection and examined formorphological signs of apoptosis The percentage of round blue apoptoticcells (mean±SD) were represented as a function of total blue cells undereach condition (n≧3). SD was less than 5%. As depicted in FIG. 4,overexpression of procaspase-3 as well as procaspase-14, but notprocaspase-8 or -10, was unable to induce any significant amount ofapoptosis. This suggests that procaspase-14, like other executionerprocaspases with small prodomains, can not undergo self activation toinduce apoptosis.

Example 4 Expression of Procaspase-14

Overexpression of procaspase-14 and procaspase-3 in bacteria.Procaspase-14 and procaspase-3 were expressed in Escherichia colipurified on Talon Ni⁺²-affinity resin (Clontech, and then analyzed bySDS-PAGE and Coomassie staining. As depicted in FIG. 5A, Lane M.molecular mass markers (kDa); lane casp-1, Talon-affinity purifiedcaspase-14; lane casp-3, Talon-affinity purified caspase-3.

Example 5 Processing of Procaspase-14

Upon expression in bacteria, all procaspases are known to autoprocess tovarious degrees to generate the mature caspase which is composed of thelarge and small subunits. The observed auto-activation in bacterialoverexpression systems is probably mediated by overexpression-inducedoligomerization. Oligomerization has been shown to induceautoactivation/processing of procaspases. Interestingly, whenprocaspase-14 was overexpressed in bacteria there was no significantprocessing of its proenzyme compared to procaspase-3 which wascompletely processes (FIG. 5A). This suggests that procaspase-14 doesnot normally process itself and it may require an upstream protease toprocess it. To test this possibility procaspase-14 was incubated withGranzyme B which is known to process several caspase proenzymes. Inaddition procaspase-14 was incubated with different purified recombinantcaspases. As shown in FIG. 5B, a significant amount of processing wasobserved when procaspase-14 was incubated with Granzyme B, caspase-10and caspase-8, but not with other caspases. Some processing was alsoobserved with recombinant caspase-14 itself, indicating that thepurified caspase-14 material contains small amount of active capase-14.These observations suggest that procaspase-14 may participate in theGranzyme B, caspase-8 and caspase-10 protease cascades.

Processing of mouse procaspase-14 by Granzyme B and purified recombinantcaspases was carried out under the following conditions: ³⁵S labeledprocaspase-14 was incubated with purified Granzyme B (14 ng/μl) or theindicated purified recombinant caspases (20 ng/μl) in ICE buffer (25 mMHepes, 1 mM EDTA, 5 mM DTT, 0.1% CHAPS, pH 7.5) at 37° C. for 1 h. Thereactions were stopped by addition of an SDS-sample buffer and then theproducts were analyzed by SDS-PAGE and autoradiography.

Since caspase-8 and -10 are initiator caspases that are activated byoligomerization of the death receptors (i.e., Fas, TRAIL-R) by theirligands or agonist antibodies, the possibility that procaspase-14 isprocessed in vivo after induction of apoptosis by anti-Fas antibody orthe cytotoxic ligand TRAIL was tested. A mammalian expression constructencoding N-terminal T7-tagged procaspase-14 was transfected intoMCF-7-FAS cells. The cells were treated 36 h after transfection withagonist anti-Fas antibody or TRAIL for 3 h. Cells were harvested andlysed by addition of SDS-sample buffer. The cellular proteins wereanalyzed by SDS-PAGE and then immunoblotted with an anti-T7HRP-conjugated monoclonal antibody (FIG. 5C, left panel) to detectprocaspase-14. The same samples were also immunoblotted with apolyclonal antibody (anti-Mch3α) that preferentially detects the proformof caspase-7 (FIG. 5C, middle panel), or a mixture of the anti-Mch3αantibody and CM-1 antibody that preferentially detects the processesfragments of caspase-7 (FIG. 5C, right panel) to detect the endogenouscaspase-7. Pro indicates the proenzyme, LS indicates the large subunitand SS indicates the small subunit. As shown in FIG. 5C, Anti-Fas andTRAIL were able to induce processing of procaspase-14 and procaspase-7as evident from the decreased intensity of their proenzyme bands. Thecleavage products of procaspase-14 were not clearly detectable probablydue to loss of the epitope tag after processing of the small prodomainof procaspase-14 at Asp7 or Asp 17. On the other hand, the cleavageproducts of procaspase-7 were clearly detectable using an antibody thatdetects the processes large subunit of procaspase-7 (FIG. 5C, rightpanel). These observations indicate that caspase-14, like caspase-7, islikely involved in the death receptor pathways.

Example 6 Cytochrome C Dependent Processing of Procaspase-14

To determine if caspase-14 was activated by a way of Apaf-1 andcaspase-9, cytochrome c dependent activation was tested. ³⁵S labeledprocaspase-3 (FIG. 6, lanes 1 & 2), procaspase-7 (FIG. 6, lanes 3 & 4),or procaspase-14 (FIG. 6, lanes 5 & 6) were incubated with S100 extractsfrom human embryonic kidney 293 cells in the absence (−) or presence (+)of cytochrome c (50 ng/μl) and dATP (1 mM) in buffer A (20 mM Hepes, 10mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, pH7.5) at 30° C. for 1 h. The reaction products were then analyzed bySDS-PAGE and autoradiography.

As indicated by FIG. 6, unlike caspase-3 or -7, no cleavage/activationof ³⁵S labeled procaspase-14 was observed in S-100 extracts activated bycytochrome c and dATP. This indicates that cytochrome c-activatedcaspase-3, -6, -7, and -9 in the S-100 cellular extract likely cannotprocess procaspase-14.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 78 <210> SEQ ID NO 1 <211> LENGTH: 850<212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:<221> NAME/KEY: modified_base <222> LOCATION: (537)<223> OTHER INFORMATION: Where n is Adenine, Cy #tosine, Guanine or      Thymine <400> SEQUENCE: 1cacgcgtccg cccacgcgtc cggtgagaca gaggcaaaac aaaggtgctg aa#agccagac     60atggagtcag agatgagtga tcctcagcca ttgcaggagg aaagatatga ta#tgtcaggt    120gcccgcctgg ccctgacgct gtgtgtcacc aaagcccggg agggttccga gg#tagacatg    180gaggccctgg aacgcatgtt ccgttacctg aaatttgaaa gcaccatgaa ga#gggatccc    240accgcccagc aatttctgga agagttggat gaatttcagc agaccataga ta#attgggaa    300gagcctgtca gctgtgcctt tgtggtactc atggcacatg gtgaggaagg cc#tcctcaag    360ggagaagatg agaagatggt cagactagaa gacctttttg aagtcttgaa ca#acaagaac    420tgcaaggccc tgagaggcaa gccaaaggtg tacatcatcc aggcttgtag ag#gagagcac    480agagaccccg gtgaggaact acgtggaaat gaggaactag gtggagatga gg#aactnggt    540ggagatgagg ttgctgtgct caagaacaac ccccaaagta tcccaaccta ta#cggatacc    600ctccacatct actccacggt agaggggtac ctctcctata gacatgacga ga#aaggctct    660ggcttcatcc agaccctgac ggatgtgttc attcataaaa aaggatccat ct#tagaactg    720acagaagaga tcacccgact tatggcaaac acggaggtga tgcaggaagg aa#aaccaagg    780aaagtgaacc ctgaagtcca aagcaccctc cggaagaagc tctatttgca at#aaaagaga    840 gggcagggat                 #                  #                   #       850 <210> SEQ ID NO 2 <211> LENGTH: 260<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 2Lys Pro Asp Met Glu Ser Glu Met Ser Asp Pr #o Gln Pro Leu Gln Glu -3      -1   1        #        5           #        10Glu Arg Tyr Asp Met Ser Gly Ala Arg Leu Al #a Leu Thr Leu Cys Val     15              #     20              #     25Thr Lys Ala Arg Glu Gly Ser Glu Val Asp Me #t Glu Ala Leu Glu Arg 30                  # 35                  # 40                  # 45Met Phe Arg Tyr Leu Lys Phe Glu Ser Thr Me #t Lys Arg Asp Pro Thr                 50  #                 55  #                 60Ala Gln Gln Phe Leu Glu Glu Leu Asp Glu Ph #e Gln Gln Thr Ile Asp             65      #             70      #             75Asn Trp Glu Glu Pro Val Ser Cys Ala Phe Va #l Val Leu Met Ala His         80          #         85          #         90Gly Glu Glu Gly Leu Leu Lys Gly Glu Asp Gl #u Lys Met Val Arg Leu     95              #    100              #    105Glu Asp Leu Phe Glu Val Leu Asn Asn Lys As #n Cys Lys Ala Leu Arg110                 1 #15                 1 #20                 1 #25Gly Lys Pro Lys Val Tyr Ile Ile Gln Ala Cy #s Arg Gly Glu His Arg                130   #               135   #               140Asp Pro Gly Glu Glu Leu Arg Gly Asn Glu Gl #u Leu Gly Gly Asp Glu            145       #           150       #           155Glu Leu Gly Gly Asp Glu Val Ala Val Leu Ly #s Asn Asn Pro Gln Ser        160           #       165           #       170Ile Pro Thr Tyr Thr Asp Thr Leu His Ile Ty #r Ser Thr Val Glu Gly    175               #   180               #   185Tyr Leu Ser Tyr Arg His Asp Glu Lys Gly Se #r Gly Phe Ile Gln Thr190                 1 #95                 2 #00                 2 #05Leu Thr Asp Val Phe Ile His Lys Lys Gly Se #r Ile Leu Glu Leu Thr                210   #               215   #               220Glu Glu Ile Thr Arg Leu Met Ala Asn Thr Gl #u Val Met Gln Glu Gly            225       #           230       #           235Lys Pro Arg Lys Val Asn Pro Glu Val Gln Se #r Thr Leu Arg Lys Lys        240           #       245           #       250 Leu Tyr Leu Gln    255 <210> SEQ ID NO 3 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 3 Gln Ala Cys Arg Gly  1               5 <210> SEQ ID NO 4 <211> LENGTH: 777 <212> TYPE: DNA<213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (49)...(774) <400> SEQUENCE: 4aggatcagac aagggtgctg agagccggga ctcacaacca aaggagaa atg #agc aat      57                    #                  #                 Met # Ser Asn                    #                  #                   #1 ccg cgg tct ttg gaa gag gag aaa tat gat at#g tca ggt gcc gcg ctg      105Pro Arg Ser Leu Glu Glu Glu Lys Tyr Asp Me #t Ser Gly Ala Ala Leu     5              #      10             #      15gcc cta ata ctg tgt gtc acc aaa gcc cgg ga#a ggt tcc gaa gaa gac      153Ala Leu Ile Leu Cys Val Thr Lys Ala Arg Gl #u Gly Ser Glu Glu Asp 20                  # 25                  # 30                  # 35ctg gat gct ctg gaa cac atg ttt cgg cag ct#g aga ttc gaa agc acc      201Leu Asp Ala Leu Glu His Met Phe Arg Gln Le #u Arg Phe Glu Ser Thr                 40  #                 45  #                 50atg aaa aga gac ccc act gcc gag caa ttc ca#g gaa gag ctg gaa aaa      249Met Lys Arg Asp Pro Thr Ala Glu Gln Phe Gl #n Glu Glu Leu Glu Lys             55      #             60      #             65ttc cag cag gcc atc gat tcc cgg gaa gat cc#c gtc agt tgt gcc ttc      297Phe Gln Gln Ala Ile Asp Ser Arg Glu Asp Pr #o Val Ser Cys Ala Phe         70          #         75          #         80gtg gta ctc atg gct cac ggg agg gaa ggc tt#c ctc aag gga gaa gat      345Val Val Leu Met Ala His Gly Arg Glu Gly Ph #e Leu Lys Gly Glu Asp     85              #     90              #     95ggg gag atg gtc aag ctg gag aat ctc ttc ga#g gcc ctg aac aac aag      393Gly Glu Met Val Lys Leu Glu Asn Leu Phe Gl #u Ala Leu Asn Asn Lys100                 1 #05                 1 #10                 1 #15aac tgc cag gcc ctg cga gct aag ccc aag gt#g tac atc ata cag gcc      441Asn Cys Gln Ala Leu Arg Ala Lys Pro Lys Va #l Tyr Ile Ile Gln Ala                120   #               125   #               130tgt cga gga gaa caa agg gac ccc ggt gaa ac#a gta ggt gga gat gag      489Cys Arg Gly Glu Gln Arg Asp Pro Gly Glu Th #r Val Gly Gly Asp Glu            135       #           140       #           145att gtg atg gtc atc aaa gac agc cca caa ac#c atc cca aca tac aca      537Ile Val Met Val Ile Lys Asp Ser Pro Gln Th #r Ile Pro Thr Tyr Thr        150           #       155           #       160gat gcc ttg cac gtt tat tcc acg gta gag gg#a tac atc gcc tac cga      585Asp Ala Leu His Val Tyr Ser Thr Val Glu Gl #y Tyr Ile Ala Tyr Arg    165               #   170               #   175cat gat cag aaa ggc tca tgc ttt atc cag ac#c ctg gtg gat gtg ttc      633His Asp Gln Lys Gly Ser Cys Phe Ile Gln Th #r Leu Val Asp Val Phe180                 1 #85                 1 #90                 1 #95acg aag agg aaa gga cat atc ttg gaa ctt ct#g aca gag gtg acc cgg      681Thr Lys Arg Lys Gly His Ile Leu Glu Leu Le #u Thr Glu Val Thr Arg                200   #               205   #               210cgg atg gca gaa gca gag ctg gtt caa gaa gg#a aaa gca agg aaa acg      729Arg Met Ala Glu Ala Glu Leu Val Gln Glu Gl #y Lys Ala Arg Lys Thr            215       #           220       #           225aac cct gaa atc caa agc acc ctc cgg aaa cg#g ctg tat ctg cag          77 #4Asn Pro Glu Ile Gln Ser Thr Leu Arg Lys Ar #g Leu Tyr Leu Gln        230           #       235           #       240tag                   #                   #                  #            777 <210> SEQ ID NO 5 <211> LENGTH: 242 <212> TYPE: PRT<213> ORGANISM: Homo sapien <400> SEQUENCE: 5Met Ser Asn Pro Arg Ser Leu Glu Glu Glu Ly #s Tyr Asp Met Ser Gly 1               5   #                10   #                15Ala Ala Leu Ala Leu Ile Leu Cys Val Thr Ly #s Ala Arg Glu Gly Ser            20       #            25       #            30Glu Glu Asp Leu Asp Ala Leu Glu His Met Ph #e Arg Gln Leu Arg Phe        35           #        40           #        45Glu Ser Thr Met Lys Arg Asp Pro Thr Ala Gl #u Gln Phe Gln Glu Glu    50               #    55               #    60Leu Glu Lys Phe Gln Gln Ala Ile Asp Ser Ar #g Glu Asp Pro Val Ser65                   #70                   #75                   #80Cys Ala Phe Val Val Leu Met Ala His Gly Ar #g Glu Gly Phe Leu Lys                85   #                90   #                95Gly Glu Asp Gly Glu Met Val Lys Leu Glu As #n Leu Phe Glu Ala Leu            100       #           105       #           110Asn Asn Lys Asn Cys Gln Ala Leu Arg Ala Ly #s Pro Lys Val Tyr Ile        115           #       120           #       125Ile Gln Ala Cys Arg Gly Glu Gln Arg Asp Pr #o Gly Glu Thr Val Gly    130               #   135               #   140Gly Asp Glu Ile Val Met Val Ile Lys Asp Se #r Pro Gln Thr Ile Pro145                 1 #50                 1 #55                 1 #60Thr Tyr Thr Asp Ala Leu His Val Tyr Ser Th #r Val Glu Gly Tyr Ile                165   #               170   #               175Ala Tyr Arg His Asp Gln Lys Gly Ser Cys Ph #e Ile Gln Thr Leu Val            180       #           185       #           190Asp Val Phe Thr Lys Arg Lys Gly His Ile Le #u Glu Leu Leu Thr Glu        195           #       200           #       205Val Thr Arg Arg Met Ala Glu Ala Glu Leu Va #l Gln Glu Gly Lys Ala    210               #   215               #   220Arg Lys Thr Asn Pro Glu Ile Gln Ser Thr Le #u Arg Lys Arg Leu Tyr225                 2 #30                 2 #35                 2 #40Leu Gln <210> SEQ ID NO 6 <211> LENGTH: 850 <212> TYPE: DNA<213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (48)...(737) <400> SEQUENCE: 6aggatcagac aagggtgctg agagccggac tcacaaccaa aggagaa atg #agc aat       56                    #                  #                Met  #Ser Asn                    #                  #                 1 ccg cgg tct ttg gaa gag gag aaa tat gat at#g tca ggt gcc cgc ctg      104Pro Arg Ser Leu Glu Glu Glu Lys Tyr Asp Me #t Ser Gly Ala Arg Leu     5              #      10             #      15gcc cta ata ctg tgt gtc acc aaa gcc cgg ga#a ggt tcc gaa gaa gac      152Ala Leu Ile Leu Cys Val Thr Lys Ala Arg Gl #u Gly Ser Glu Glu Asp 20                  # 25                  # 30                  # 35ctg gat gct ctg gaa cac atg ttt cgg cag ct#g aga ttc gaa agc acc      200Leu Asp Ala Leu Glu His Met Phe Arg Gln Le #u Arg Phe Glu Ser Thr                 40  #                 45  #                 50atg aaa aga gac ccc act gcc gag caa ttc ca#g gaa gag ctg gaa aaa      248Met Lys Arg Asp Pro Thr Ala Glu Gln Phe Gl #n Glu Glu Leu Glu Lys             55      #             60      #             65ttc cag cag gcc atc gat tcc cgg gaa gat cc#c gtc agt tgt gcc ttc      296Phe Gln Gln Ala Ile Asp Ser Arg Glu Asp Pr #o Val Ser Cys Ala Phe         70          #         75          #         80gtg gta ctc atg gct cac ggg agg gaa ggc tt#c ctc aag gga gaa gat      344Val Val Leu Met Ala His Gly Arg Glu Gly Ph #e Leu Lys Gly Glu Asp     85              #     90              #     95ggg gag atg gtc aag ctg gag aat ctc ttc ga#g gcc ctg aac aac aag      392Gly Glu Met Val Lys Leu Glu Asn Leu Phe Gl #u Ala Leu Asn Asn Lys100                 1 #05                 1 #10                 1 #15aac tgc cag gcc ctg cga gct aag ccc aag gt#g tac atc ata cag gcc      440Asn Cys Gln Ala Leu Arg Ala Lys Pro Lys Va #l Tyr Ile Ile Gln Ala                120   #               125   #               130tgt cga gga gaa caa agg gac ccc ggt gaa ac#a gta ggt gga gat gag      488Cys Arg Gly Glu Gln Arg Asp Pro Gly Glu Th #r Val Gly Gly Asp Glu            135       #           140       #           145att gtg atg gtc atc aaa gac agc cca caa ac#c atc cca aca tac aca      536Ile Val Met Val Ile Lys Asp Ser Pro Gln Th #r Ile Pro Thr Tyr Thr        150           #       155           #       160gat gcc ttg cac gtt tat tcc acg gta gag gg#a ccc acg ccc ttc cag      584Asp Ala Leu His Val Tyr Ser Thr Val Glu Gl #y Pro Thr Pro Phe Gln    165               #   170               #   175gat ccc ctc tac cta ccc tct gaa gct ccc cc#g aac cca cct ctc tgg      632Asp Pro Leu Tyr Leu Pro Ser Glu Ala Pro Pr #o Asn Pro Pro Leu Trp180                 1 #85                 1 #90                 1 #95aat tcc cag gat aca tcg cct acc gac atg at#c aga aag gct cat gct      680Asn Ser Gln Asp Thr Ser Pro Thr Asp Met Il #e Arg Lys Ala His Ala                200   #               205   #               210tta tcc aga ccc tgg tgg atg tgt tca cga ag#a gga aag gac ata tct      728Leu Ser Arg Pro Trp Trp Met Cys Ser Arg Ar #g Gly Lys Asp Ile Ser            215       #           220       #           225tgg aac ttc tgacagaggt gacccggcgg atggcagaag cagagctgg#t              777 Trp Asn Phe         230tcaagaagga aaagcaagga aaacgaaccc tgaaatccaa agcaccctcc gg#aaacggct    837 gtatctgcag tag               #                  #                   #     850 <210> SEQ ID NO 7 <211> LENGTH: 230<212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 7Met Ser Asn Pro Arg Ser Leu Glu Glu Glu Ly #s Tyr Asp Met Ser Gly 1               5   #                10   #                15Ala Arg Leu Ala Leu Ile Leu Cys Val Thr Ly #s Ala Arg Glu Gly Ser            20       #            25       #            30Glu Glu Asp Leu Asp Ala Leu Glu His Met Ph #e Arg Gln Leu Arg Phe        35           #        40           #        45Glu Ser Thr Met Lys Arg Asp Pro Thr Ala Gl #u Gln Phe Gln Glu Glu    50               #    55               #    60Leu Glu Lys Phe Gln Gln Ala Ile Asp Ser Ar #g Glu Asp Pro Val Ser65                   #70                   #75                   #80Cys Ala Phe Val Val Leu Met Ala His Gly Ar #g Glu Gly Phe Leu Lys                85   #                90   #                95Gly Glu Asp Gly Glu Met Val Lys Leu Glu As #n Leu Phe Glu Ala Leu            100       #           105       #           110Asn Asn Lys Asn Cys Gln Ala Leu Arg Ala Ly #s Pro Lys Val Tyr Ile        115           #       120           #       125Ile Gln Ala Cys Arg Gly Glu Gln Arg Asp Pr #o Gly Glu Thr Val Gly    130               #   135               #   140Gly Asp Glu Ile Val Met Val Ile Lys Asp Se #r Pro Gln Thr Ile Pro145                 1 #50                 1 #55                 1 #60Thr Tyr Thr Asp Ala Leu His Val Tyr Ser Th #r Val Glu Gly Pro Thr                165   #               170   #               175Pro Phe Gln Asp Pro Leu Tyr Leu Pro Ser Gl #u Ala Pro Pro Asn Pro            180       #           185       #           190Pro Leu Trp Asn Ser Gln Asp Thr Ser Pro Th #r Asp Met Ile Arg Lys        195           #       200           #       205Ala His Ala Leu Ser Arg Pro Trp Trp Met Cy #s Ser Arg Arg Gly Lys    210               #   215               #   220Asp Ile Ser Trp Asn Phe 225                 2 #30 <210> SEQ ID NO 8<211> LENGTH: 693 <212> TYPE: DNA <213> ORGANISM: Homo sapien<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (49)...(690)<400> SEQUENCE: 8aggatcagac aagggtgctg agagccggga ctcacaacca aaggagaa atg #agc aat      57                    #                  #                 Met # Ser Asn                    #                  #                   #1 ccg cgg tct ttg gaa gag gag aaa tat gat at#g tca ggt gcc cgc ctg      105Pro Arg Ser Leu Glu Glu Glu Lys Tyr Asp Me #t Ser Gly Ala Arg Leu     5              #      10             #      15gcc cta ata ctg tgt gtc acc aaa gcc cgg ga#a ggt tcc gaa gaa gaa      153Ala Leu Ile Leu Cys Val Thr Lys Ala Arg Gl #u Gly Ser Glu Glu Glu 20                  # 25                  # 30                  # 35gag ctg gaa aaa ttc cag cag gcc atc gat tc#c cgg gaa gat ccc gtc      201Glu Leu Glu Lys Phe Gln Gln Ala Ile Asp Se #r Arg Glu Asp Pro Val                 40  #                 45  #                 50agt tgt gcc ttc gtg gta ctc atg gct cac gg#g agg gaa ggc ttc ctc      249Ser Cys Ala Phe Val Val Leu Met Ala His Gl #y Arg Glu Gly Phe Leu             55      #             60      #             65aag gga gaa gat ggg gag atg gtc aag ctg ga#g aat ctc ttc gag gcc      297Lys Gly Glu Asp Gly Glu Met Val Lys Leu Gl #u Asn Leu Phe Glu Ala         70          #         75          #         80ctg aac aac aag aac tgc cag gcc ctg cga gc#t aag ccc aag gtg tac      345Leu Asn Asn Lys Asn Cys Gln Ala Leu Arg Al #a Lys Pro Lys Val Tyr     85              #     90              #     95atc ata cag gcc tgt cga gga gaa caa agg ga#c ccc ggt gaa aca gta      393Ile Ile Gln Ala Cys Arg Gly Glu Gln Arg As #p Pro Gly Glu Thr Val100                 1 #05                 1 #10                 1 #15ggt gga gat gag att gtg atg gtc atc aaa ga#c agc cca caa acc atc      441Gly Gly Asp Glu Ile Val Met Val Ile Lys As #p Ser Pro Gln Thr Ile                120   #               125   #               130cca aca tac aca gat gcc ttg cac gtt tat tc#c acg gta gag gga tac      489Pro Thr Tyr Thr Asp Ala Leu His Val Tyr Se #r Thr Val Glu Gly Tyr            135       #           140       #           145atc gcc tac cga cat gat cag aaa ggc tca tg#c ttt atc cag acc ctg      537Ile Ala Tyr Arg His Asp Gln Lys Gly Ser Cy #s Phe Ile Gln Thr Leu        150           #       155           #       160gtg gat gtg ttc acg aag agg aaa gga cat at#c ttg gaa ctt ctg aca      585Val Asp Val Phe Thr Lys Arg Lys Gly His Il #e Leu Glu Leu Leu Thr    165               #   170               #   175gag gtg acc cgg cgg atg gca gaa gca gag ct#g gtt caa gaa gga aaa      633Glu Val Thr Arg Arg Met Ala Glu Ala Glu Le #u Val Gln Glu Gly Lys180                 1 #85                 1 #90                 1 #95gca agg aaa acg aac cct gaa atc caa agc ac#c ctc cgg aaa cgg ctg      681Ala Arg Lys Thr Asn Pro Glu Ile Gln Ser Th #r Leu Arg Lys Arg Leu                200   #               205   #               210tat ctg cag tag             #                   #                  #      693 Tyr Leu Gln <210> SEQ ID NO 9 <211> LENGTH: 214<212> TYPE: PRT <213> ORGANISM: Homo sapien <400> SEQUENCE: 9Met Ser Asn Pro Arg Ser Leu Glu Glu Glu Ly #s Tyr Asp Met Ser Gly 1               5   #                10   #                15Ala Arg Leu Ala Leu Ile Leu Cys Val Thr Ly #s Ala Arg Glu Gly Ser            20       #            25       #            30Glu Glu Glu Glu Leu Glu Lys Phe Gln Gln Al #a Ile Asp Ser Arg Glu        35           #        40           #        45Asp Pro Val Ser Cys Ala Phe Val Val Leu Me #t Ala His Gly Arg Glu    50               #    55               #    60Gly Phe Leu Lys Gly Glu Asp Gly Glu Met Va #l Lys Leu Glu Asn Leu65                   #70                   #75                   #80Phe Glu Ala Leu Asn Asn Lys Asn Cys Gln Al #a Leu Arg Ala Lys Pro                85   #                90   #                95Lys Val Tyr Ile Ile Gln Ala Cys Arg Gly Gl #u Gln Arg Asp Pro Gly            100       #           105       #           110Glu Thr Val Gly Gly Asp Glu Ile Val Met Va #l Ile Lys Asp Ser Pro        115           #       120           #       125Gln Thr Ile Pro Thr Tyr Thr Asp Ala Leu Hi #s Val Tyr Ser Thr Val    130               #   135               #   140Glu Gly Tyr Ile Ala Tyr Arg His Asp Gln Ly #s Gly Ser Cys Phe Ile145                 1 #50                 1 #55                 1 #60Gln Thr Leu Val Asp Val Phe Thr Lys Arg Ly #s Gly His Ile Leu Glu                165   #               170   #               175Leu Leu Thr Glu Val Thr Arg Arg Met Ala Gl #u Ala Glu Leu Val Gln            180       #           185       #           190Glu Gly Lys Ala Arg Lys Thr Asn Pro Glu Il #e Gln Ser Thr Leu Arg        195           #       200           #       205Lys Arg Leu Tyr Leu Gln     210 <210> SEQ ID NO 10 <211> LENGTH: 22<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 10Met Ala Glu Asn Lys His Pro Asp Lys Pro Le #u Lys Val Leu Glu Gln  1               5  #                 10  #                 15Leu Gly Lys Glu Val Leu              20 <210> SEQ ID NO 11<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 11 Thr Glu Tyr Leu Glu Lys Leu Val Gln Ser As#n Val Leu Lys Leu Lys   1               5  #                 10 #                 15 Glu Glu Asp Lys Gln Lys Phe Asn Asn Ala Gl#u Arg Ser Asp Lys Arg              20      #             25     #             30 Trp Val Phe Val          35 <210> SEQ ID NO 12<211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 12 Asp Ala Met Lys Lys Lys His Ser Lys Val Gl#y Glu Met Leu Leu Gln   1               5  #                 10 #                 15 Thr Phe Phe Ser Val Asp Pro Gly Ser His Hi#s Gly Glu Ala Asn Leu              20      #             25     #             30 Glu Met Glu Glu Pro Glu Glu Ser Leu Asn Th#r Leu Lys Leu Cys Ser          35          #         40         #         45 Pro Glu Glu Phe Thr Arg Leu Cys Arg Glu Ly#s Thr Gln Glu Ile Tyr      50              #     55             #     60 Pro Ile Lys Glu Ala Asn  65                  # 70<210> SEQ ID NO 13 <211> LENGTH: 62 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 13Gly Arg Thr Arg Lys Ala Leu Ile Ile Cys As #n Thr Glu Phe Lys His  1               5  #                 10  #                 15Leu Ser Leu Arg Tyr Gly Ala Asn Phe Asp Il #e Ile Gly Met Lys Gly             20      #             25      #             30Leu Leu Glu Asp Leu Gly Tyr Asp Val Val Va #l Lys Glu Glu Leu Thr         35          #         40          #         45Ala Glu Gly Met Glu Ser Glu Met Asp Lys Ph #e Ala Ala Leu     50              #     55              #     60 <210> SEQ ID NO 14<211> LENGTH: 94 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 14 Ser Glu His Gln Thr Ser Asp Ser Thr Phe Le#u Val Leu Met Ser His   1               5  #                 10 #                 15 Gly Thr Leu His Gly Ile Cys Gly Thr Met Hi#s Ser Glu Lys Thr Pro              20      #             25     #             30 Asp Val Leu Gln Tyr Asp Thr Ile Tyr Gln Il#e Phe Asn Asn Cys His          35          #         40         #         45 Cys Pro Gly Leu Arg Asp Lys Pro Lys Val Il#e Ile Val Gln Ala Cys      50              #     55             #     60 Arg Gly Gly Asn Ser Gly Glu Met Trp Ile Ar#g Glu Ser Ser Lys Pro  65                  # 70                 # 75                  # 80 Gln Leu Cys Arg Gly Val Asp Leu Pro Arg As#n Met Glu Ala                  85  #                 90<210> SEQ ID NO 15 <211> LENGTH: 89 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 15Asp Ala Val Lys Leu Ser His Val Glu Lys As #p Phe Ile Ala Phe Tyr  1               5  #                 10  #                 15Ser Thr Thr Pro His His Leu Ser Tyr Arg As #p Lys Thr Gly Gly Ser             20      #             25      #             30Tyr Phe Ile Thr Arg Leu Ile Ser Cys Phe Ar #g Lys His Ala Cys Ser         35          #         40          #         45Cys His Leu Phe Asp Ile Phe Leu Lys Val Gl #n Gln Ser Phe Glu Lys     50              #     55              #     60Ala Ser Ile His Ser Gln Met Pro Thr Ile As #p Arg Ala Thr Leu Thr 65                  # 70                  # 75                  # 80Arg Tyr Phe Tyr Leu Phe Pro Gly Asn                  85<210> SEQ ID NO 16 <211> LENGTH: 172 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 16Met Ala Ala Arg Arg Thr His Glu Arg Asp Pr #o Ile Tyr Lys Ile Lys  1               5  #                 10  #                 15Gly Leu Ala Lys Asp Met Leu Asp Gly Val Ph #e Asp Asp Leu Val Glu             20      #             25      #             30Lys Asn Val Leu Asn Gly Asp Glu Leu Leu Ly #s Ile Gly Glu Ser Ala         35          #         40          #         45Ser Phe Ile Leu Asn Lys Ala Glu Asn Leu Va #l Glu Asn Phe Leu Glu     50              #     55              #     60Lys Thr Asp Met Ala Gly Lys Ile Phe Ala Gl #y His Ile Ala Asn Ser 65                  # 70                  # 75                  # 80Gln Glu Gln Leu Ser Leu Gln Phe Ser Asn As #p Glu Asp Asp Gly Pro                 85  #                 90  #                 95Gln Lys Ile Cys Thr Pro Ser Ser Pro Ser Gl #u Ser Lys Arg Lys Val            100       #           105       #           110Glu Asp Asp Glu Met Glu Val Asn Ala Gly Le #u Ala His Glu Ser His        115           #       120           #       125Leu Met Leu Thr Ala Pro His Gly Leu Gln Se #r Ser Glu Val Gln Asp    130               #   135               #   140Thr Leu Lys Leu Cys Pro Arg Asp Gln Phe Cy #s Lys Ile Lys Thr Glu145                 1 #50                 1 #55                 1 #60Arg Ala Lys Glu Ile Tyr Pro Val Met Glu Ly #s Glu                 165  #               170 <210> SEQ ID NO 17 <211> LENGTH: 62 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 17Gly Arg Thr Arg Leu Ala Leu Ile Ile Cys As #n Lys Lys Phe Asp Tyr  1               5  #                 10  #                 15Leu Phe Asp Arg Asp Asn Ala Asp Thr Asp Il #e Leu Asn Met Gln Glu             20      #             25      #             30Leu Leu Glu Asn Leu Gly Tyr Ser Val Val Le #u Lys Glu Asn Leu Thr         35          #         40          #         45Ala Gln Glu Met Glu Thr Glu Leu Met Gln Ph #e Ala Gly Arg     50              #     55              #     60 <210> SEQ ID NO 18<211> LENGTH: 74 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 18 Pro Glu His Gln Ser Ser Asp Ser Thr Phe Le#u Val Phe Met Ser His   1               5  #                 10 #                 15 Gly Ile Leu Glu Gly Ile Cys Gly Val Lys Hi#s Arg Asn Lys Lys Pro              20      #             25     #             30 Asp Val Leu His Asp Asp Thr Ile Phe Lys Il#e Phe Asn Asn Ser Asn          35          #         40         #         45 Cys Arg Ser Leu Arg Asn Lys Pro Lys Ile Le#u Ile Met Gln Ala Cys      50              #     55             #     60 Arg Gly Arg Tyr Asn Gly Thr Ile Trp Val  65                 # 70 <210> SEQ ID NO 19 <211> LENGTH: 21 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 19Ser Thr Asn Lys Gly Ile Ala Thr Ala Asp Th #r Asp Glu Glu Arg Val  1               5  #                 10  #                 15Leu Ser Cys Lys Trp              20 <210> SEQ ID NO 20 <211> LENGTH: 90<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 20Asn Asn Ser Ile Thr Lys Ala His Val Glu Th #r Asp Phe Ile Ala Phe  1               5  #                 10  #                 15Lys Ser Ser Thr Pro His Asn Ile Ser Trp Ar #g Val Gly Lys Thr Gly             20      #             25      #             30Ser Leu Phe Ile Ser Lys Leu Ile Asp Cys Ph #e Lys Lys Tyr Cys Trp         35          #         40          #         45Cys Tyr His Leu Glu Glu Ile Phe Arg Lys Va #l Gln His Ser Phe Glu     50              #     55              #     60Val Pro Gly Glu Leu Thr Gln Met Pro Thr Il #e Glu Arg Val Ser Met 65                  # 70                  # 75                  # 80Thr Arg Tyr Phe Tyr Leu Phe Pro Gly Asn                  85 #                 90 <210> SEQ ID NO 21 <211> LENGTH: 119<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 21Met Ala Asp Lys Ile Leu Arg Ala Lys Arg Ly #s Gln Phe Ile Asn Ser  1               5  #                 10  #                 15Val Ser Ile Gly Thr Ile Asn Gly Leu Leu As #p Glu Leu Leu Glu Lys             20      #             25      #             30Arg Val Leu Asn Gln Glu Glu Met Asp Lys Il #e Lys Leu Ala Asn Ile         35          #         40          #         45Thr Ala Met Asp Lys Ala Arg Asp Leu Cys As #p His Val Ser Lys Lys     50              #     55              #     60Gly Pro Gln Ala Ser Gln Ile Phe Ile Thr Ty #r Ile Cys Asn Glu Asp 65                  # 70                  # 75                  # 80Cys Tyr Leu Ala Gly Ile Leu Glu Leu Gln Se #r Ala Pro Ser Ala Glu                 85  #                 90  #                 95Thr Phe Val Ala Thr Glu Asp Ser Lys Gly Gl #y His Pro Ser Ser Ser            100       #           105       #           110Glu Thr Lys Glu Glu Gln Asn         115 <210> SEQ ID NO 22<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 22 Lys Glu Asp Gly Thr Phe Pro Gly Leu Thr Gl #y  1               5  #                 10 <210> SEQ ID NO 23<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 23 Thr Leu Lys Phe Cys Pro Leu Glu Lys Ala Gl#n Lys Leu Trp Lys Glu   1               5  #                 10 #                 15 Asn Pro Ser Glu Ile Tyr Pro Ile Met Asn Th #r Thr             20      #             25 <210> SEQ ID NO 24<211> LENGTH: 62 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 24 Thr Arg Thr Arg Leu Ala Leu Ile Ile Cys As#n Thr Glu Phe Gln His   1               5  #                 10 #                 15 Leu Ser Pro Arg Val Gly Ala Gln Val Asp Le#u Arg Glu Met Lys Leu              20      #             25     #             30 Leu Leu Glu Asp Leu Gly Tyr Thr Val Lys Va#l Lys Glu Asn Leu Thr          35          #         40         #         45 Ala Leu Glu Met Val Lys Glu Val Lys Glu Ph #e Ala Ala Cys     50              #     55              #     60 <210> SEQ ID NO 25<211> LENGTH: 77 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 25 Pro Glu His Lys Thr Ser Asp Ser Thr Phe Le#u Val Phe Met Ser His   1               5  #                 10 #                 15 Gly Ile Gln Glu Gly Ile Cys Gly Thr Thr Ty#r Ser Asn Glu Val Ser              20      #             25     #             30 Asp Ile Leu Lys Val Asp Thr Ile Phe Gln Me#t Met Asn Thr Leu Lys          35          #         40         #         45 Cys Pro Ser Leu Lys Asp Lys Pro Lys Val Il#e Ile Ile Gln Ala Cys      50              #     55             #     60 Arg Gly Glu Lys Gln Gly Val Val Leu Leu Ly #s Asp Ser 65                  # 70                  # 75 <210> SEQ ID NO 26<211> LENGTH: 105 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 26 Val Arg Asp Ser Glu Glu Asp Phe Leu Thr As#p Ala Ile Phe Glu Asp   1               5  #                 10 #                 15 Asp Gly Ile Lys Lys Ala His Ile Glu Lys As#p Phe Ile Ala Phe Cys              20      #             25     #             30 Ser Ser Thr Pro Asp Asn Val Ser Trp Arg Hi#s Pro Val Arg Gly Ser          35          #         40         #         45 Leu Phe Ile Glu Ser Leu Ile Lys His Met Ly#s Glu Tyr Ala Trp Ser      50              #     55             #     60 Cys Asp Leu Glu Asp Ile Phe Arg Lys Val Ar#g Phe Ser Phe Glu Gln  65                  # 70                 # 75                  # 80 Pro Glu Phe Arg Leu Gln Met Pro Thr Ala As#p Arg Val Thr Leu Thr                  85  #                 90 #                 95 Lys Arg Phe Tyr Leu Phe Pro Gly His            100       #           105 <210> SEQ ID NO 27<211> LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 27 Met Glu Asn Asn Lys Thr Ser Val Asp Ser Ly#s Ser Ile Asn Asn Phe   1               5  #                 10 #                 15 Glu Val Lys Thr Ile His Gly Ser Lys Ser Va#l Asp Ser Gly Ile Tyr              20      #             25     #             30 Leu Asp Ser Ser Tyr Lys Met Asp Tyr Pro Gl#u Met Gly Ile Cys Ile          35          #         40         #         45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser     50              #     55 <210> SEQ ID NO 28 <211> LENGTH: 47<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 28Thr Gly Met Ser Ser Arg Ser Gly Thr Asp Va #l Asp Ala Ala Asn Leu  1               5  #                 10  #                 15Arg Glu Thr Phe Met Gly Leu Lys Tyr Cys Va #l Arg Asn Lys Asn Asp             20      #             25      #             30Leu Thr Arg Glu Asp Ile Leu Glu Leu Met As #p Ser Val Ser Lys         35          #         40          #         45<210> SEQ ID NO 29 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 29Glu Asp His Ser Lys Arg Ser Ser Phe Val Cy #s Val Ile Leu Ser His  1               5  #                 10  #                 15Gly Asp Glu Gly Val Ile Tyr Gly Thr Asn Gl #y Pro              20     #             25 <210> SEQ ID NO 30 <211> LENGTH: 39 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 30Val Glu Leu Lys Lys Leu Thr Ser Phe Phe Ar #g Gly Asp Tyr Cys Arg  1               5  #                 10  #                 15Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Il #e Gln Ala Cys Arg Gly             20      #             25      #             30Thr Glu Leu Asp Cys Gly Ile          35 <210> SEQ ID NO 31<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 31 Glu Thr Asp Ser   1 <210> SEQ ID NO 32<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 32 Thr Asp Glu Glu Met Ala Cys   1               5<210> SEQ ID NO 33 <211> LENGTH: 42 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 33Gln Lys Ile Pro Val Glu Ala Asp Phe Leu Ty #r Ala Tyr Ser Thr Ala  1               5  #                 10  #                 15Pro Gly Tyr Tyr Ser Trp Arg Asn Ser Lys As #p Gly Ser Trp Phe Ile             20      #             25      #             30Gln Ser Leu Cys Ser Met Leu Lys Leu Tyr          35         #         40 <210> SEQ ID NO 34 <211> LENGTH: 51 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 34Ala His Lys Leu Glu Phe Met His Ile Leu Th #r Arg Val Asn Arg Lys  1               5  #                 10  #                 15Val Ala Thr Glu Phe Glu Ser Phe Ser Leu As #p Ser Thr Phe His Ala             20      #             25      #             30Lys Lys Gln Ile Pro Cys Ile Val Ser Met Le #u Thr Lys Glu Leu Tyr         35          #         40          #         45 Phe Tyr His     50 <210> SEQ ID NO 35 <211> LENGTH: 81 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 35Met Thr Asp Asp Gln Asp Cys Ala Ala Glu Le #u Glu Lys Val Asp Ser  1               5  #                 10  #                 15Ser Ser Glu Asp Gly Val Asp Ala Lys Pro As #p Arg Ser Ser Ile Ile             20      #             25      #             30Ser Ser Ile Leu Leu Lys Lys Lys Arg Asn Al #a Ser Ala Gly Pro Val         35          #         40          #         45Arg Thr Gly Arg Asp Arg Val Pro Thr Tyr Le #u Tyr Arg Met Asp Phe     50              #     55              #     60Gln Lys Met Gly Lys Cys Ile Ile Ile Asn As #n Lys Asn Phe Asp Lys 65                  # 70                  # 75                  # 80Ala <210> SEQ ID NO 36 <211> LENGTH: 47 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 36Thr Gly Met Asp Val Arg Asn Gly Thr Asp Ly #s Asp Ala Gly Ala Leu  1               5  #                 10  #                 15Phe Lys Cys Phe Gln Asn Leu Gly Phe Glu Va #l Thr Val His Asn Asp             20      #             25      #             30Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Ar #g Lys Ala Ser Glu         35          #         40          #         45<210> SEQ ID NO 37 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 37Glu Asp His Ser Asn Ser Ala Cys Phe Ala Cy #s Val Leu Leu Ser His  1               5  #                 10  #                 15Gly Glu Glu Asp Leu Ile Tyr Gly Lys Asp Gl #y Val              20     #             25 <210> SEQ ID NO 38 <211> LENGTH: 39 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 38Thr Pro Ile Lys Asp Leu Thr Ala His Phe Ar #g Gly Asp Arg Cys Lys  1               5  #                 10  #                 15Thr Leu Leu Glu Lys Pro Lys Leu Phe Phe Il #e Gln Ala Cys Arg Gly             20      #             25      #             30Thr Glu Leu Asp Asp Gly Ile          35 <210> SEQ ID NO 39<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 39 Gln Ala Asp Ser   1 <210> SEQ ID NO 40<211> LENGTH: 52 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 40 Pro Ile Asn Asp Ile Asp Ala Asn Pro Arg As#n Lys Ile Pro Val Glu   1               5  #                 10 #                 15 Ala Asp Phe Leu Phe Ala Tyr Ser Thr Val Pr#o Gly Tyr Tyr Ser Trp              20      #             25     #             30 Arg Asn Pro Gly Lys Gly Ser Trp Phe Val Gl#n Ala Leu Cys Ser Ile          35          #         40         #         45 Leu Asn Glu His      50 <210> SEQ ID NO 41 <211> LENGTH: 51<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 41Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Th #r Arg Val Asn Asp Arg  1               5  #                 10  #                 15Val Ala Arg His Phe Glu Ser Gln Ser Asp As #p Pro Arg Phe Asn Glu             20      #             25      #             30Lys Lys Gln Ile Pro Cys Met Val Ser Met Le #u Thr Lys Glu Leu Tyr         35          #         40          #         45 Phe Ser Arg     50 <210> SEQ ID NO 42 <211> LENGTH: 41 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 42Met Thr Glu Thr Asp Gly Phe Tyr Lys Ser Ar #g Glu Val Phe Asp Pro  1               5  #                 10  #                 15Ala Glu Gln Tyr Lys Met Asp His Lys Arg Ar #g Gly Val Ala Leu Ile             20      #             25      #             30Phe Asn His Glu Arg Phe Phe Trp His          35          #         40<210> SEQ ID NO 43 <211> LENGTH: 47 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 43Leu Thr Leu Pro Glu Arg Arg Gly Thr Asn Al #a Asp Arg Asp Asn Leu  1               5  #                 10  #                 15Thr Arg Arg Phe Ser Asp Leu Gly Phe Glu Va #l Lys Cys Phe Asn Asp             20      #             25      #             30Leu Arg Ala Glu Glu Leu Leu Leu Lys Ile Hi #s Glu Val Ser Thr         35          #         40          #         45<210> SEQ ID NO 44 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 44Ser Ser His Ile Asp Ala Asp Cys Phe Ile Cy #s Val Phe Leu Ser His  1               5  #                 10  #                 15Gly Glu Gly Asn His Val Tyr Ala Tyr Asp Al #a Lys              20     #             25 <210> SEQ ID NO 45 <211> LENGTH: 51 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 45Ile Glu Ile Gln Thr Leu Thr Gly Leu Phe Ly #s Gly Asp Lys Cys Gln  1               5  #                 10  #                 15Ser Leu Val Gly Lys Pro Lys Ile Phe Ile Il #e Gln Ala Cys Arg Gly             20      #             25      #             30Ser Gln His Asp Val Pro Val Val Pro Leu As #p Met Val Asp His Gln         35          #         40          #         45 Thr Asp Lys     50 <210> SEQ ID NO 46 <211> LENGTH: 52 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 46Asn Val Thr Gln Val Asp Ala Ala Ser Val Ty #r Thr Leu Pro Ala Gly  1               5  #                 10  #                 15Ala Asp Phe Leu Met Cys Tyr Ser Val Ala Gl #u Gly Tyr Tyr Ser His             20      #             25      #             30Arg Glu Thr Val Asn Gly Ser Trp Tyr Ile Gl #n Asp Leu Cys Glu Met         35          #         40          #         45 Leu Ala Arg Tyr     50 <210> SEQ ID NO 47 <211> LENGTH: 55 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 47Gly Ser Ser Leu Glu Phe Thr Glu Leu Leu Th #r Leu Val Asn Arg Lys  1               5  #                 10  #                 15Val Ser Gln Arg Arg Val Asp Phe Cys Lys As #p Pro Asp Ala Ile Gly             20      #             25      #             30Lys Lys Gln Val Pro Cys Phe Ala Ser Met Le #u Thr Lys Lys Leu His         35          #         40          #         45Phe Cys Pro Lys Pro Ser Lys      50              #     55<210> SEQ ID NO 48 <211> LENGTH: 250 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 48Met Asp Phe Gln Ser Cys Leu Asp Ala Ile Al #a Glu Glu Leu Gly Ser  1               5  #                 10  #                 15Glu Asp Leu Ala Ala Leu Lys Phe Leu Cys Le #u Asp Tyr Ile Pro His             20      #             25      #             30Lys Lys Leu Glu Thr Ile Glu Asp Ala Gln Ly #s Leu Phe Leu Arg Leu         35          #         40          #         45Arg Glu Lys Gly Met Leu Glu Glu Gly Asn Le #u Ser Phe Leu Lys Glu     50              #     55              #     60Leu Leu Phe His Ile Ser Arg Trp Asp Leu Le #u Val Asn Phe Leu Asp 65                  # 70                  # 75                  # 80Cys Asn Arg Glu Glu Met Val Arg Glu Leu Ar #g Asp Pro Arg Gln Cys                 85  #                 90  #                 95Pro Arg Phe Leu Pro Tyr Arg Ser Cys Ser Ph #e Arg Leu Ser Glu Glu            100       #           105       #           110Val Ser Glu Leu Glu Leu Arg Ser Phe Lys Ph #e Leu Leu Asn Asn Glu        115           #       120           #       125Ile Pro Lys Cys Lys Leu Glu Asp Asp Leu Se #r Leu Leu Glu Ile Phe    130               #   135               #   140Val Glu Met Glu Lys Arg Thr Met Leu Ala Gl #u Asn Asn Leu Glu Thr145                 1 #50                 1 #55                 1 #60Leu Lys Ser Ile Cys Asp Gln Val Asn Lys Se #r Leu Leu Gly Lys Ile                165   #               170   #               175Glu Asp Tyr Glu Arg Ser Ser Thr Glu Arg Ar #g Met Ser Leu Glu Gly            180       #           185       #           190Arg Glu Glu Leu Pro Pro Ser Val Leu Asp Gl #u Met Ser Leu Lys Met        195           #       200           #       205Ala Glu Leu Cys Asp Ser Pro Arg Glu Gln As #p Ser Glu Ser Arg Thr    210               #   215               #   220Ser Asp Lys Val Tyr Gln Met Lys Asn Lys Pr #o Arg Gly Tyr Cys Leu225                 2 #30                 2 #35                 2 #40Ile Ile Asn Asn His Asp Phe Ser Lys Ala                 245  #               250 <210> SEQ ID NO 49 <211> LENGTH: 54 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 49Arg Glu Asp Ile Thr Gln Leu Arg Lys Met Ly #s Asp Arg Lys Gly Thr  1               5  #                 10  #                 15Asp Cys Asp Lys Glu Ala Leu Ser Lys Thr Ph #e Lys Glu Leu His Phe             20      #             25      #             30Glu Ile Val Ser Tyr Asp Asp Cys Thr Ala As #n Glu Ile His Glu Ile         35          #         40          #         45Leu Glu Gly Tyr Gln Ser      50 <210> SEQ ID NO 50 <211> LENGTH: 28<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 50Ala Asp His Lys Asn Lys Asp Cys Phe Ile Cy #s Cys Ile Leu Ser His  1               5  #                 10  #                 15Gly Asp Lys Gly Val Val Tyr Gly Thr Asp Gl #y Lys              20     #             25 <210> SEQ ID NO 51 <211> LENGTH: 52 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 51Glu Ala Ser Ile Tyr Asp Leu Thr Ser Tyr Ph #e Thr Gly Ser Lys Cys  1               5  #                 10  #                 15Pro Ser Leu Ser Gly Lys Pro Lys Ile Phe Ph #e Ile Gln Ala Cys Arg             20      #             25      #             30Gly Ser Asn Phe Gln Lys Gly Val Pro Asp Gl #u Ala Gly Phe Glu Gln         35          #         40          #         45 Gln Asn His Thr     50 <210> SEQ ID NO 52 <211> LENGTH: 47 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 52Ser Ser His Lys Asn Tyr Ile Pro Asp Glu Al #a Asp Phe Leu Leu Gly  1               5  #                 10  #                 15Met Ala Thr Val Leu Met Cys Val Ser Tyr Ar #g Asp Pro Val Asn Gly             20      #             25      #             30Thr Trp Tyr Ile Gln Ser Leu Cys Gln Ser Le #u Arg Glu Arg Cys         35          #         40          #         45<210> SEQ ID NO 53 <211> LENGTH: 19 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 53Pro Gln Gly Asp Asp Ile Leu Ser Ile Leu Th #r Gly Val Asn Tyr Asp  1               5  #                 10  #                 15Val Ser Asn <210> SEQ ID NO 54 <211> LENGTH: 22 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 54Lys Asp Asp Arg Arg Asn Lys Gly Lys Gln Me #t Pro Gln Pro Thr Phe  1               5  #                 10  #                 15Thr Leu Arg Lys Lys Leu              20 <210> SEQ ID NO 55<211> LENGTH: 260 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 55 Met Ala Ala Pro Ser Gly Arg Ser Gln Ser Se#r Leu His Arg Lys Gly   1               5  #                 10 #                 15 Leu Met Ala Ala Asp Arg Arg Ser Arg Ile Le#u Ala Val Cys Gly Met              20      #             25     #             30 His Pro Asp His Gln Glu Thr Leu Lys Lys As#n Arg Val Val Leu Ala          35          #         40         #         45 Lys Gln Leu Leu Leu Ser Glu Leu Leu Glu Hi#s Leu Leu Glu Lys Asp      50              #     55             #     60 Ile Ile Thr Leu Glu Met Arg Glu Leu Ile Gl#n Ala Lys Gly Gly Ser  65                  # 70                 # 75                  # 80 Phe Ser Gln Asn Val Glu Leu Leu Asn Leu Le#u Pro Lys Arg Gly Pro                  85  #                 90 #                 95 Gln Ala Phe Asp Ala Phe Cys Glu Ala Leu Ar#g Glu Thr Arg Gln Gly             100       #           105      #           110 His Leu Glu Asp Leu Leu Leu Thr Thr Leu Se#r Asp Ile Gln His Val         115           #       120          #       125 Leu Pro Pro Leu Ser Cys Asp Tyr Asp Thr Se#r Leu Pro Phe Ser Val     130               #   135              #   140 Cys Glu Ser Cys Pro Pro His Lys Gln Leu Ar#g Leu Ser Thr Asp Ala 145                 1 #50                 1#55                 1 #60 Thr Glu His Ser Leu Asp Asn Gly Asp Gly Pr#o Pro Cys Leu Leu Val                 165   #               170  #               175 Lys Pro Cys Thr Pro Glu Phe Tyr Gln Ala Hi#s Tyr Gln Leu Ala Tyr             180       #           185      #           190 Arg Leu Gln Ser Gln Pro Arg Gly Leu Ala Le#u Val Leu Ser Asn Val         195           #       200          #       205 His Phe Thr Gly Glu Lys Asp Leu Glu Phe Ar#g Ser Gly Gly Asp Val     210               #   215              #   220 Asp His Thr Thr Leu Val Thr Leu Phe Lys Le#u Leu Gly Tyr Asn Val 225                 2 #30                 2#35                 2 #40 His Val Leu His Asp Gln Thr Ala Gln Glu Me#t Gln Glu Lys Leu Gln                 245   #               250  #               255 Asn Phe Ala Gln             260 <210> SEQ ID NO 56<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 56 Leu Pro Ala His Arg Val Thr Asp Ser Val Cy #s  1               5  #                 10 <210> SEQ ID NO 57<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 57 Val Ala Leu Leu Ser His Gly Val Glu Gly Gl#y Ile Tyr Gly Val Asp   1               5  #                 10 #                 15 Gly Lys <210> SEQ ID NO 58 <211> LENGTH: 56<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 58Leu Leu Gln Leu Gln Glu Val Phe Arg Leu Ph #e Asp Asn Ala Asn Cys  1               5  #                 10  #                 15Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Ph #e Ile Gln Ala Cys Arg             20      #             25      #             30Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gl #n Asp Gly Lys Asn His         35          #         40          #         45Thr Gln Ser Pro Gly Cys Glu Glu      50              #     55<210> SEQ ID NO 59 <211> LENGTH: 53 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 59Ser Asp Ala Gly Lys Glu Glu Leu Met Lys Me #t Arg Leu Pro Thr Arg  1               5  #                 10  #                 15Ser Asp Met Ile Cys Gly Tyr Ala Cys Leu Ly #s Gly Asn Ala Ala Met             20      #             25      #             30Arg Asn Thr Lys Arg Gly Ser Trp Tyr Ile Gl #u Ala Leu Thr Gln Val         35          #         40          #         45Phe Ser Glu Arg Ala      50 <210> SEQ ID NO 60 <211> LENGTH: 18<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 60Asp Met His Val Ala Asp Met Leu Val Lys Va #l Asn Ala Leu Ile Lys  1               5  #                 10  #                 15 Glu Arg<210> SEQ ID NO 61 <211> LENGTH: 35 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 61Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Ar #g Cys Lys Glu Met Ser  1               5  #                 10  #                 15Glu Tyr Cys Ser Thr Leu Cys Gln Gln Leu Ty #r Leu Phe Pro Gly Tyr             20      #             25      #             30 Pro Pro Thr         35 <210> SEQ ID NO 62 <211> LENGTH: 31 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 62Met Glu Ser Glu Met Ser Asp Pro Gln Pro Le #u Gln Glu Glu Arg Tyr  1               5  #                 10  #                 15Asp Met Ser Gly Ala Arg Leu Ala Leu Thr Le #u Cys Val Thr Lys             20      #             25      #             30<210> SEQ ID NO 63 <211> LENGTH: 74 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 63Ala Arg Glu Gly Ser Glu Val Asp Met Glu Al #a Leu Glu Arg Met Phe  1               5  #                 10  #                 15Arg Tyr Leu Lys Phe Glu Ser Thr Met Lys Ar #g Asp Pro Thr Ala Gln             20      #             25      #             30Gln Phe Leu Glu Glu Leu Asp Glu Phe Gln Gl #n Thr Ile Asp Asn Trp         35          #         40          #         45Glu Glu Pro Val Ser Cys Ala Phe Val Val Le #u Met Ala His Gly Glu     50              #     55              #     60Glu Gly Leu Leu Lys Gly Glu Asp Glu Lys  65                  # 70<210> SEQ ID NO 64 <211> LENGTH: 56 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 64Met Val Arg Leu Glu Asp Leu Phe Glu Val Le #u Asn Asn Lys Asn Cys  1               5  #                 10  #                 15Lys Ala Leu Arg Gly Lys Pro Lys Val Tyr Il #e Ile Gln Ala Cys Arg             20      #             25      #             30Gly Glu His Arg Asp Pro Gly Glu Glu Leu Ar #g Gly Asn Glu Glu Leu         35          #         40          #         45Gly Gly Asp Glu Glu Leu Gly Gly      50              #     55<210> SEQ ID NO 65 <211> LENGTH: 53 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 65Asp Glu Val Ala Val Leu Lys Asn Asn Pro Gl #n Ser Ile Pro Thr Tyr  1               5  #                 10  #                 15Thr Asp Thr Leu His Ile Tyr Ser Thr Val Gl #u Gly Tyr Leu Ser Tyr             20      #             25      #             30Arg His Asp Glu Lys Gly Ser Gly Phe Ile Gl #n Thr Leu Thr Asp Val         35          #         40          #         45Phe Ile His Lys Lys      50 <210> SEQ ID NO 66 <211> LENGTH: 15<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 66Ile Leu Glu Leu Thr Glu Glu Ile Thr Arg Le #u Met Ala Asn Thr  1               5  #                 10  #                 15<210> SEQ ID NO 67 <211> LENGTH: 8 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 67Glu Val Met Gln Glu Gly Lys Pro   1               5 <210> SEQ ID NO 68<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Mus musculus<400> SEQUENCE: 68 Arg Lys Val Asn Pro Glu Val Gln Ser Thr Le#u Arg Lys Lys Leu Tyr   1               5  #                 10 #                 15 Leu Gln <210> SEQ ID NO 69 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer derived from mouse # caspase-14 cDNA<400> SEQUENCE: 69 atatgatatg tcaggtgccc g           #                   #                   #21 <210> SEQ ID NO 70<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer derived from mouse# caspase-14 cDNA <400> SEQUENCE: 70 ttccggaggg tgctttgga             #                   #                   # 19 <210> SEQ ID NO 71<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer derived from human# caspase-14 cDNA <400> SEQUENCE: 71 cctgtatgat gtacaccttg g           #                   #                   #21 <210> SEQ ID NO 72<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer derived from human# caspase-14 cDNA <400> SEQUENCE: 72 agagattctc cagcttgac             #                   #                   # 19 <210> SEQ ID NO 73<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer derived from human# caspase-14 cDNA <400> SEQUENCE: 73 atcttctccc ttgaggaag             #                   #                   # 19 <210> SEQ ID NO 74<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer dereived from huma#n caspase-14 cDNA <400> SEQUENCE: 74 atatgatatg tcaggtgccc g           #                   #                   #21 <210> SEQ ID NO 75<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer derived from human# caspase-14 cDNA <400> SEQUENCE: 75 caaggtgtac atcatacagg            #                   #                   # 20 <210> SEQ ID NO 76<211> LENGTH: 850 <212> TYPE: DNA <213> ORGANISM: Mus musculus<220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (537)<223> OTHER INFORMATION: Where n is Adenine, Cy #tosine, Guanin or      Thymidine <400> SEQUENCE: 76gtgcgcaggc gggtgcgcag gccactctgt ctccgttttg tttccacgac tt#tcggtctg     60tacctcagtc tctactcact aggagtcggt aacgtcctcc tttctatact at#acagtcca    120cgggcggacc gggactgcga cacacagtgg tttcgggccc tcccaaggct cc#atctgtac    180ctccgggacc ttgcgtacaa ggcaatggac tttaaacttt cgtggtactt ct#ccctaggg    240tggcgggtcg ttaaagacct tctcaaccta cttaaagtcg tctggtatct at#taaccctt    300ctcggacagt cgacacggaa acaccatgag taccgtgtac cactccttcc gg#aggagttc    360cctcttctac tcttctacca gtctgatctt ctggaaaaac ttcagaactt gt#tgttcttg    420acgttccggg actctccgtt cggtttccac atgtagtagg tccgaacatc tc#ctctcgtg    480tctctggggc cactccttga tgcaccttta ctccttgatc cacctctact cc#ttgancca    540cctctactcc aacgacacga gttcttgttg ggggtttcat agggttggat at#gcctatgg    600gaggtgtaga tgaggtgcca tctccccatg gagaggatat ctgtactgct ct#ttccgaga    660ccgaagtagg tctgggactg cctacacaag taagtatttt ttcctaggta ga#atcttgac    720tgtcttctct agtgggctga ataccgtttg tgcctccact acgtccttcc tt#ttggttcc    780tttcacttgg gacttcaggt ttcgtgggag gccttcttcg agataaacgt ta#ttttctct    840 cccgtcccta                 #                  #                   #       850 <210> SEQ ID NO 77 <211> LENGTH: 16<212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 77His Ala Ser Ala His Ala Ser Gly Glu Thr Gl #u Ala Lys Gln Arg Cys  1               5  #                 10  #                 15<210> SEQ ID NO 78 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 78 Lys Arg Gly Gln Gly  1               5

We claim:
 1. An isolated monoclonal antibody or an antigen binding fragment thereof specific for a caspase-14 polypeptide, wherein said polypeptide comprises SEQ ID NO:2.
 2. The antibody of claim 1, wherein said antigen binding fragment comprises an Fv portion of an antibody.
 3. An isolated cell expressing the antibody of claim
 1. 4. An isolated monoclonal antibody or antigen binding fragment thereof specific for a caspase-14 polypeptide, wherein said polypeptide comprises SEQ ID NO:5.
 5. The antibody of claim 4, wherein said antigen binding fragment comprises an Fv portion of an antibody. 